Combination therapies for treating cancer

ABSTRACT

Provided are methods of treating cancer that comprise administering a polypeptide (e.g. a fusion polypeptide) that comprises a SIRPα D1 domain variant and an Fc domain variant in combination with at least one chemotherapy agent and/or at least one therapeutic antibody. Also provided are related kits.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication 62/941,390, filed Nov. 27, 2019; U.S. ProvisionalApplication 63/022,998, filed May 11, 2020; U.S. Provisional Application63/030,686, filed May 27, 2020; U.S. Provisional Application 63/106,225,filed Oct. 27, 2020; and U.S. Provisional Application 63/109,044, filedNov. 3, 2020, the contents of each of which are incorporated herein byreference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 757972001100SEQLIST.TXT,date recorded: Nov. 25, 2020, size: 333 KB).

FIELD OF THE INVENTION

The present invention relates to methods of treating cancer thatcomprise administering an agent that blocks the interaction between CD47(e.g., hCD47) and SIRPα (e.g., hSIRPα) in conjunction with achemotherapy agent and at least one additional anti-cancer agent and/orat least one additional mode of cancer therapy.

BACKGROUND

Many cancers have a poor prognosis, even when treated with availabletherapeutics. There is a need in the art for new treatments to provideadditional therapeutic options and improve outcomes for patents.

Tumor cells manipulate the myeloid compartment to evade the anti-tumorhost immune response (Gabrilovich et al., Nat Rev Immunol (2012)12(4):253-68). For example, while CD47 expressed on the surface ofnormal cells binds SIRPα on macrophages and provides a “don't eat me”signal, tumor cells have also been found to overexpress CD47 to evadethe macrophage component of immune surveillance (Oldenborg, ISRN Hematol(2013) 614619).

Macrophage-mediated destruction of cancer cells requires both thedisruption of “don't eat me” signals (e.g., CD47-SIRPα) and theactivation of “eat me” signals. Neither component alone is sufficient totrigger maximal phagocytic reaction against tumor cells. As describedabove, CD47 provides a fundamental “don't eat me” signal through itsinteraction with SIRPα on macrophages. The pro-phagocytic “eat me”signal can be provided to the same macrophages by binding to theiractivating Fc gamma receptors. For example, the pro-phagocytic “eat me”signal can be provided by binding of anti-tumor antibodies to Fcreceptors on macrophages.

All references cited herein, including patent applications, patentpublications, and UniProtKB/Swiss-Prot Accession numbers are hereinincorporated by reference in their entirety, as if each individualreference were specifically and individually indicated to beincorporated by reference.

BRIEF SUMMARY

Provided is a method of treating cancer in an individual, comprisingadministering to the individual an effective amount of: (a) apolypeptide comprising a SIRPα D1 domain variant and an Fc domainvariant, and (b) a Bcl-2 inhibitor; wherein the SIRPα D1 domain variantcomprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85;wherein the Fc domain variant is (i) a human IgG1 Fc region comprisingL234A, L235A, G237A, and N297A mutations, wherein numbering is accordingto the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S,P331S, and N297A mutations, wherein numbering is according to the EUindex of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P,F234V, L235A, and delG236 mutations, wherein numbering is according tothe EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P,E233P, F234V, L235A, delG236, and N297A mutations, wherein numbering isaccording to the EU index of Kabat. In some embodiments, the cancer isleukemia, multiple myeloma, or non-Hodgkin's lymphoma. In someembodiments, the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma(DLBCL), mantle cell lymphoma (MCL), or follicular lymphoma (FL). Insome embodiments, the leukemia is acute lymphoblastic leukemia (ALL),chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL),Chronic myeloid leukemia (CIVIL), acute myeloid leukemia (AML), ormyelodysplastic syndrome (MDS). In some embodiments, the leukemia isacute lymphoblastic leukemia. In some embodiments, the Bcl-2 inhibitoris venetoclax, ABT-737, navitoclax, BCL201, or AZD-0466. In someembodiments, the Bcl-2 inhibitor is venetoclax.

Also provided is a method of treating cancer in an individual,comprising administering to the individual an effective amount of: (a) apolypeptide comprising a SIRPα D1 domain variant and an Fc domainvariant, and (b) a platinum-based chemotherapy agent; wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 orSEQ ID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fcregion comprising L234A, L235A, G237A, and N297A mutations, whereinnumbering is according to the EU index of Kabat; (ii) a human IgG2 Fcregion comprising A330S, P331S, and N297A mutations, wherein numberingis according to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat. Insome embodiments, the cancer is a solid tumor. In some embodiments, thesolid tumor is colon cancer, lung cancer, head and neck cancer,esophageal cancer, breast cancer, bladder cancer, ovarian cancer,cervical cancer, testicular cancer, endometrial cancer, liver cancer,gastric cancer, gastroesophageal junction cancer, brain tumor,mesothelioma, or neuroblastoma. In some embodiments, the colon cancer iscolon carcinoma. In some embodiments, the platinum-based chemotherapyagent is carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatintetranitrate, phenanthriplatin, picoplatin, or satraplatin. In someembodiments, the platinum-based chemotherapy agent is cisplatin orcarboplatin.

Also provided is a method of treating cancer in an individual,comprising administering to the individual an effective amount of: (a) apolypeptide comprising a SIRPα D1 domain variant and an Fc domainvariant, (b) a PD-1 inhibitor, (c) an antimetabolite, and (d) aplatinum-based chemotherapy agent; wherein the SIRPα D1 domain variantcomprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85;wherein the Fc domain variant is (i) a human IgG1 Fc region comprisingL234A, L235A, G237A, and N297A mutations, wherein numbering is accordingto the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S,P331S, and N297A mutations, wherein numbering is according to the EUindex of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P,F234V, L235A, and delG236 mutations, wherein numbering is according tothe EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P,E233P, F234V, L235A, delG236, and N297A mutations, wherein numbering isaccording to the EU index of Kabat, wherein the cancer is head and necksquamous cell carcinoma (HNSCC), and wherein the individual has notreceived prior treatment for HNSCC. In some embodiments, the polypeptidecomprising the SIRPα D1 domain variant and the Fc domain variant isadministered at a dose of 10 mg/kg once a week (qw). In someembodiments, the polypeptide comprising the SIRPα D1 domain variant andthe Fc domain variant is administered at a dose of 15 mg/kg once a week(qw).

In some embodiments, the HNSCC is advanced and/or metastatic HNSCC. Insome embodiments, the PD-1 inhibitor is an anti-PD-1 antibody, e.g.,pembrolizumab, nivolumab, pidilizumab, cemiplimab, or BMS-936559. Insome embodiments, the anti-PD-1 antibody is pembrolizumab. In someembodiments, the antimetabolite is 5-fluorouracil, 6-mercaptopurine,capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine,hydroxycarbamide, methotrexate, pemetrexed, phototrexate. In someembodiments, the antimetabolite is 5-fluorouracil. In some embodiments,the platinum-based chemotherapy agent is carboplatin, cisplatin,oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin,picoplatin, or satraplatin. In some embodiments, the platinum-basedchemotherapy agent is cisplatin or carboplatin.

In another aspect, provided is a method of treating cancer in anindividual, comprising administering to the individual an effectiveamount of: (a) a polypeptide comprising a SIRPα D1 domain variant and anFc domain variant, (b) an anti-HER2 antibody, and (c) an anti-PD-L1antibody (e.g., an anti-PD-L1 antagonist antibody); wherein the SIRPα D1domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat. Insome embodiments, the cancer is solid tumor. In some embodiments, thesolid tumor is colon cancer, lung cancer, head and neck cancer,esophageal cancer, breast cancer, bladder cancer, ovarian cancer,cervical cancer, testicular cancer, endometrial cancer, liver cancer,gastric cancer, gastroesophageal junction cancer, brain tumor,mesothelioma, or neuroblastoma. In some embodiments, the solid tumor isHER2⁺ solid tumor. In some embodiments, the solid tumor is colon cancer(e.g., HER2⁺ colon cancer). In some embodiments, the anti-HER2 antibodyis trastuzumab. In some embodiments, the anti-PD-L1 antibody isatezolizumab, avelumab, or durvalumab.

In some embodiments, provided is a method of treating cancer in anindividual, comprising administering to the individual an effectiveamount of: (a) a polypeptide comprising a SIRPα D1 domain variant and anFc domain variant, (b) an anti-HER2 antibody, (c) an anti-VEGF2antibody, and (d) paclitaxel; wherein the SIRPα D1 domain variantcomprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85;wherein the Fc domain variant is (i) a human IgG1 Fc region comprisingL234A, L235A, G237A, and N297A mutations, wherein numbering is accordingto the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S,P331S, and N297A mutations, wherein numbering is according to the EUindex of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P,F234V, L235A, and delG236 mutations, wherein numbering is according tothe EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P,E233P, F234V, L235A, delG236, and N297A mutations, wherein numbering isaccording to the EU index of Kabat, wherein the cancer is gastric canceror gastroesophageal junction (GEJ) cancer, and wherein the individualhas received at least one prior therapy for the gastric or the GEJcancer. In some embodiments, the gastric cancer or GEJ cancer is aHER2-overexpressing (e.g., HER2⁺) gastric cancer or aHER2-overexpressing GEJ cancer. In some embodiments, the individual hasreceived prior therapy with an anti-HER2 antibody, with an anti-HER2antibody and a fluoropyrimidine, or with an anti HER2 antibody and aplatinum-based chemotherapy agent. In some embodiments, the anti-HER2antibody is trastuzumab. In some embodiments, the anti-VEGF antibody isramucirumab. In some embodiments, the polypeptide comprising the SIRPαD1 domain variant and the Fc domain variant is administered at a dose of10 mg/kg once a week (qw). In some embodiments, the polypeptidecomprising the SIRPα D1 domain variant and the Fc domain variant isadministered at a dose of 15 mg/kg once a week (qw).

Also provided is a method of treating cancer in an individual,comprising administering to the individual an effective amount of (a) apolypeptide comprising a SIRPα D1 domain variant and an Fc domainvariant, and (b) an anti-TROP2 antibody; wherein the SIRPα D1 domainvariant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat. Insome embodiments, the cancer is solid tumor, gastric cancer,nasopharyngeal cancer, gallbladder cancer, cervical cancer, extranodalNK/T cell lymphoma, lung cancer, laryngeal squamous cell cancer, coloncancer, Hilar Cholangiocarcinoma, pancreatic cancer, squamous cellcarcinoma of the oral cavity, endometrioid endometrial carcinoma, orovarian carcinoma.

In some embodiments of any of the methods described herein, the SIRPα D1domain variant comprises the amino acid sequence of SEQ ID NO: 85. Insome embodiments, the SIRPα D1 domain variant comprises the amino acidsequence of SEQ ID NO: 81. In some embodiments, the Fc domain variant isa human IgG1 Fc region comprising L234A, L235A, G237A, and N297Amutations, wherein numbering is according to the EU index of Kabat. Insome embodiments, the Fc domain variant comprises the amino acidsequence of SEQ ID NO: 91. In some embodiments, the polypeptidecomprising a SIRPα D1 domain variant and an Fc domain variant comprisesthe amino acid sequence of SEQ ID NO: 136. In some embodiments, thepolypeptide comprising a SIRPα D1 domain variant and an Fc domainvariant comprises the amino acid sequence of SEQ ID NO: 135. In someembodiments, the polypeptide comprising a SIRPα D1 domain variant and anFc domain variant forms a homodimer. In some embodiments, the individualis a human.

In another aspect, provided is a kit comprising a polypeptide comprisinga SIRPα D1 domain variant and an Fc domain variant in a pharmaceuticallyacceptable carrier, for use in combination with a Bcl-2 inhibitor fortreating cancer in an individual in need, wherein the SIRPα D1 domainvariant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat; andwherein the kit comprises instructions for administering the polypeptidecomprising a SIRPα D1 domain variant and an Fc domain variant incombination with the Bcl-2 inhibitor to the individual in need thereof.In some embodiments, the cancer is leukemia, multiple myeloma, ornon-Hodgkin's lymphoma. In some embodiments, the Bcl-2 inhibitor isvenetoclax.

Also provided is a kit comprising a polypeptide comprising a SIRPα D1domain variant and an Fc domain variant in a pharmaceutically acceptablecarrier, for use in combination with a platinum-based chemotherapy agentfor treating cancer in an individual in need thereof, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 orSEQ ID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fcregion comprising L234A, L235A, G237A, and N297A mutations, whereinnumbering is according to the EU index of Kabat; (ii) a human IgG2 Fcregion comprising A330S, P331S, and N297A mutations, wherein numberingis according to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat; andwherein the kit comprises instructions for administering the polypeptidecomprising a SIRPα D1 domain variant and an Fc domain variant incombination with the chemotherapy agent to the individual in needthereof. In some embodiments, the cancer is solid tumor. In someembodiments, the solid tumor is colon cancer, colon carcinoma, lungcancer, head and neck cancer, esophageal cancer, breast cancer, bladdercancer, ovarian cancer, cervical cancer, testicular cancer, endometrialcancer, liver cancer, gastric cancer, brain tumor, mesothelioma, orneuroblastoma. In some embodiments, the platinum-based chemotherapyagent is cisplatin or carboplatin.

In some embodiments, provided is a kit comprising a polypeptidecomprising a SIRPα D1 domain variant and an Fc domain variant in apharmaceutically acceptable carrier, for use in combination with a PD-1inhibitor, an antimetabolite, and a platinum-based chemotherapy agentfor treating cancer in an individual in need thereof, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 orSEQ ID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fcregion comprising L234A, L235A, G237A, and N297A mutations, whereinnumbering is according to the EU index of Kabat; (ii) a human IgG2 Fcregion comprising A330S, P331S, and N297A mutations, wherein numberingis according to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat, andwherein the kit comprises instructions for administering the polypeptidecomprising a SIRPα D1 domain variant and an Fc domain variant incombination with the anti-PD-1 antibody, the antimetabolite, and theplatinum-based chemotherapy agent to an individual with head and necksquamous cell carcinoma (HNSCC) who has not received prior treatment forHNSCC. In some embodiments, the PD-1 inhibitor is pembrolizumab. In someembodiments, the antimetabolite is 5-fluorouracil. In some embodiments,the platinum-based chemotherapy agent is cisplatin or carboplatin.

In some embodiments, provided is a kit comprising a polypeptidecomprising a SIRPα D1 domain variant and an Fc domain variant in apharmaceutically acceptable carrier, for use in combination with ananti-HER2 antibody, an anti-VEGFR2 antibody, and paclitaxel; wherein theSIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a human IgG1Fc region comprising L234A, L235A, G237A, and N297A mutations, whereinnumbering is according to the EU index of Kabat; (ii) a human IgG2 Fcregion comprising A330S, P331S, and N297A mutations, wherein numberingis according to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat, andwherein the kit comprises instructions for administering the polypeptidecomprising a SIRPα D1 domain variant and an Fc domain variant incombination with the anti-HER2 antibody, the anti-VEGFR2 antibody, andthe paclitaxel to an individual with gastric cancer or gastroesophagealjunction (GEJ) cancer who has received at least one prior therapy forthe gastric or the GEJ cancer. In some embodiments, the gastric canceror GEJ cancer is HER2⁺ gastric cancer or HER2⁺ GEJ cancer. In someembodiments, the anti-HER2 antibody is trastuzumab. In some embodiments,the anti-VEGFR2 antibody is ramucirumab. In some embodiments, theindividual received prior therapy (or therapies) with an anti-HER2antibody (e.g., trastuzumab) and/or a fluoropyrimidine, and/or aplatinum-based chemotherapeutic agent. In some embodiments, the gastriccancer or GEJ cancer in the individual progressed during or after priortherapy (or therapies) comprising anti-HER2 antibody (e.g., trastuzumab)and/or a fluoropyrimidine, and/or a platinum-based chemotherapeuticagent. In some embodiments, the individual failed (e.g., relapsed afteror did not respond to) prior therapy (or therapies) comprising anti-HER2antibody (e.g., trastuzumab) and/or a fluoropyrimidine, and/or aplatinum-based chemotherapeutic agent. In some embodiments, the priortherapy (or therapies) comprised an anti-HER2 antibody and afluoropyrimidine (e.g., administered during the same line of therapy orduring different lines of therapy). In some embodiments, the priortherapy (or therapies) comprised an anti-HER2 antibody and aplatinum-based chemotherapy agent (e.g., administered during the sameline of therapy or during different lines of therapy)

Also provided is a kit comprising a polypeptide comprising a SIRPα D1domain variant and an Fc domain variant in a pharmaceutically acceptablecarrier, for use in combination with an anti-TROP2 antibody for treatingcancer in an individual in need thereof, wherein the SIRPα D1 domainvariant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat; andwherein the kit comprises instructions for administering the polypeptidecomprising a SIRPα D1 domain variant and an Fc domain variant incombination with the anti-TROP2 antibody to the individual in needthereof. In some embodiments, the cancer is solid tumor, gastric cancer,nasopharyngeal cancer, gallbladder cancer, cervical cancer, extranodalNK/T cell lymphoma, lung cancer, laryngeal squamous cell cancer, coloncancer, Hilar Cholangiocarcinoma, pancreatic cancer, squamous cellcarcinoma of the oral cavity, endometrioid endometrial carcinoma, orovarian carcinoma.

Also provided is a kit comprising a polypeptide comprising a SIRPα D1domain variant and an Fc domain variant in a pharmaceutically acceptablecarrier, for use in combination with an anti-HER2 antibody and ananti-PD-L1 antibody (e.g., an anti PD-L1 antagonist antibody) fortreating cancer in an individual in need thereof, wherein the SIRPα D1domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat; andwherein the kit comprises instructions for administering the polypeptidecomprising a SIRPα D1 domain variant and an Fc domain variant incombination with the anti-HER2 antibody and the anti-PD-L1 antibody(e.g., an anti PD-L1 antagonist antibody) to the individual in needthereof. In some embodiments, the cancer is colon cancer. In someembodiments, the colon cancer is HER2⁺ colon cancer. In someembodiments, the anti-HER2 antibody is trastuzumab. In some embodiments,the anti-PD-L1 antibody is atezolizumab, avelumab, or durvalumab.

In some embodiments of the kits, the SIRPα D1 domain variant comprisesthe amino acid sequence of SEQ ID NO: 85. In some embodiments, the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81. Insome embodiments, the Fc domain variant is a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat. In some embodiments, the Fcdomain variant comprises the amino acid sequence of SEQ ID NO: 91. Insome embodiments, the polypeptide comprising a SIRPα D1 domain variantand an Fc domain variant comprises the amino acid sequence of SEQ ID NO:136. In some embodiments, the polypeptide comprising a SIRPα D1 domainvariant and an Fc domain variant comprises the amino acid sequence ofSEQ ID NO: 135. In some embodiments, the polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant forms a homodimer. In someembodiments, the individual is a human.

DESCRIPTION OF THE FIGURES

FIG. 1A provides tumor volumes (mm³) in NOD-SCID female mice injectedwith RS4; 11 leukemia cells following treatment with Drug A, venetoclax,venetoclax/Drug A combination, or vehicle (PBS) at the indicated timespost implant. Dashed arrows indicate dosing of venetoclax (250 μg) byoral gavage 2 times total, 3 days apart. Dotted arrows indicate dosingof Drug A (10 mg/kg) 4 times total, 3-4 days apart. SEM=standard errorof the mean; TF=tumor free.

FIG. 1B provides tumor volumes (mm³) at the indicated times post implantin NOD-SCID female mice injected with RS4; 11 leukemia cells that hadreceived treatment venetoclax, and which were subsequently re-treatedwith single agent venetoclax or with a venetoclax/Drug A combination.

FIGS. 2A-2D provide the tumor volume (mm³) and body weight of BALB/cfemale mice injected with CT26 tumor cells following treatment with DrugA, Cisplatin, Cisplatin/Drug A combination, or vehicle (PBS) at theindicated times post implantation. FIG. 2A provides the mean tumorvolumes (+/−SEM) for the indicated treatments. Dashed arrows indicatedosing of Cisplatin (two 5 mg/kg doses given 10 days apart). Dottedarrows indicate dosing of Drug A (two 30 mg/kg doses given 10 daysapart). Both drugs were administered intraperitoneally. Mice treatedwith both agents were dosed with Drug A one day post treatment withcisplatin. FIG. 2B provides the mean percent change in body weight fromday 7 (D7) in mice treated according to the regimens shown in FIG. 2A.FIG. 2C provides the mean tumor volumes (+/−SEM) for the indicatedtreatments. Dashed arrows indicate dosing of Cisplatin (one 10 mg/kgdose). Dotted arrows indicate dosing of Drug A (two 30 mg/kg doses given10 days apart). Both drugs were administered intraperitoneally. Micetreated with both agents were dosed with Drug A one day post treatmentwith cisplatin. FIG. 2D provides the mean percent change in body weightfrom day 7 (D7) in mice treated according to the regimens shown in FIG.2C.

FIG. 3 provides the results of experiments that were performed todetermine the effect of Drug A in combination with an anti-TROP2antibody on the phagocytosis of CFSE-labeled DLD-1 tumor cells by humanmonocyte-derived macrophages.

FIG. 4 provides the results of experiments that were performed todetermine the effect of Drug A in combination with (a) an anti-HER2antibody, (b) an anti-PD-L1 antibody, or (c) an anti-HER2 antibody andan anti-PD-L1 on tumor growth in a MC38 m/h colon cancer model.

FIG. 5A provides the results of experiments that were performed toassess the effects of the addition of Drug A, venetoclax, or both Drug Aand venetoclax in the phagocytosis of HL60 cells by macrophages in an invitro assay. FIG. 5B provides the results of experiments that wereperformed to assess the effects of the addition of Drug A, venetoclax,or both Drug A and venetoclax in the phagocytosis of OCI-AML3 cells bymacrophages in an in vitro assay.

FIG. 6A provides the results of experiments that were performed toassess the effects of Drug A or Drug C on CD8⁺ dendritic cellactivation. FIG. 6B provides the results of experiments that wereperformed to assess the effects of Drug A or Drug C on CD8⁻ dendriticcell activation.

FIG. 7A provides the results of experiments that were performed toassess the effects of Drug A or Drug B on CD8⁺ dendritic cellactivation. FIG. 7B provides the results of experiments that wereperformed to assess the effects of Drug A or Drug B on CD8⁻ dendriticcell activation.

FIG. 8A provides the results of experiments that were performed toassess the binding of Drug A, F59/magrolimab, TTI-621, and TTI-622 tohCD47. FIG. 8B provides the results of quantitative experiments thatwere performed to assess the effect of Drug A, F59/magrolimab, TTI-621,and TTI-622 on SIRPα signaling.

DETAILED DESCRIPTION

The following description sets forth exemplary methods, parameters andthe like. It should be recognized, however, that such description is notintended as a limitation on the scope of the present disclosure but isinstead provided as a description of exemplary embodiments.

Definitions

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, up to 10%, up to 5%, or up to 1% of a given value.Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude, preferablywithin 5-fold, and more preferably within 2-fold, of a value. Whereparticular values are described in the application and claims, unlessotherwise stated the term “about” meaning within an acceptable errorrange for the particular value should be assumed.

The terminology used herein is for the purpose of describing particularcases only and is not intended to be limiting. As used herein, thesingular forms “a”, “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.Furthermore, to the extent that the terms “including”, “includes”,“having”, “has”, “with”, or variants thereof are used in either thedetailed description or the claims, such terms are intended to beinclusive in a manner similar to the term “comprising.”

As used herein, the terms “treatment”, “treating”, and the like, referto administering an agent, or carrying out a procedure, for the purposesof obtaining an effect. In some embodiments, the effect is prophylacticin terms of completely or partially preventing a disease or symptomthereof. In some embodiments, the effect is therapeutic in terms ofaffecting a partial or complete cure for a disease or symptoms of thedisease.

As used herein, the term “antibody” refers to intact antibodies;antibody fragments, provided that they exhibit the desired biologicalactivity (e.g. epitope binding); monoclonal antibodies; polyclonalantibodies; monospecific antibodies; multi-specific antibodies (e.g.,bispecific antibodies); and antibody-like proteins.

As used herein, the term “antibody variable domain” refers to theportions of the light and heavy chains of an antibody that include aminoacid sequences of complementary determining regions (CDRs, e.g., CDR L1,CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3) and framework regions (FRs).

As used herein, the term “linker” refers to a linkage between twoelements, e.g., protein domains. In some embodiments, a linker can be acovalent bond or a spacer. The term “spacer” refers to a moiety (e.g., apolyethylene glycol (PEG) polymer) or an amino acid sequence (e.g., a1-200 amino acid sequence) occurring between two polypeptides orpolypeptide domains to provide space or flexibility (or both space andflexibility) between the two polypeptides or polypeptide domains. Insome embodiments, an amino acid spacer is part of the primary sequenceof a polypeptide (e.g., joined to the spaced polypeptides or polypeptidedomains via the polypeptide backbone).

As used herein, the term “effective amount” refers to an amount of apolypeptide or a pharmaceutical composition containing a polypeptidedescribed herein, e.g., a polypeptide having a SIRPα D1 domain orvariant thereof, that is sufficient and effective in achieving a desiredtherapeutic effect in treating a patient having a disease, such as acancer, e.g., solid tumor or hematological cancer. In some embodiments,an effective amount of polypeptide will avoid adverse side effects.

As used herein, the term “pharmaceutical composition” refers to amedicinal or pharmaceutical formulation that includes an activeingredient as well as excipients or diluents (or both excipients anddiluents) and enables the active ingredient to be administered bysuitable methods of administration. In some embodiments, thepharmaceutical compositions disclosed herein include pharmaceuticallyacceptable components that are compatible with the polypeptide. In someembodiments, the pharmaceutical composition is in tablet or capsule formfor oral administration or in aqueous form for intravenous orsubcutaneous administration, for example by injection.

As used herein, the terms “subject,” “individual,” and “patient” areused interchangeably to refer to a vertebrate, for example, a mammal.Mammals include, but are not limited to, murines, simians, humans, farmanimals, sport animals, and pets. Tissues, cells, and their progeny of abiological entity obtained in vivo or cultured in vitro are alsoencompassed. None of the terms entail supervision of a medicalprofessional.

As used herein, the term “affinity” or “binding affinity” refers to thestrength of the binding interaction between two molecules. Generally,binding affinity refers to the strength of the sum total of non-covalentinteractions between a molecule and its binding partner, such as a SIRPαD1 domain variant and CD47. Unless indicated otherwise, binding affinityrefers to intrinsic binding affinity, which reflects a 1:1 interactionbetween members of a binding pair. The binding affinity between twomolecules is commonly described by the dissociation constant (K_(D)) orthe association constant (KA). Two molecules that have low bindingaffinity for each other generally bind slowly, tend to dissociateeasily, and exhibit a large K_(D). Two molecules that have high affinityfor each other generally bind readily, tend to remain bound longer, andexhibit a small K_(D). In some embodiments, the K_(D) of two interactingmolecules is determined using known methods and techniques, e.g.,surface plasmon resonance (SPR). K_(D) can be calculated as the ratio ofkoff/kon.

As used herein, the term “K_(D) less than” refers to a numericallysmaller K_(D) value and an increasing binding affinity relative to therecited K_(D) value. As used herein, the term “K_(D) greater than”refers to a numerically larger K_(D) value and a decreasing bindingaffinity relative to the recited K_(D) value.

As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during, or after administration of the other treatment modalityto the individual.

Overview

Provided herein are methods of treating cancer in an individual (e.g., ahuman individual) that comprises administering to the individual aneffective amount of (a) an agent that blocks the interaction betweenCD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) and (b) a chemotherapy agent(e.g., at least one chemotherapy agent, such as at least two, at leastthree, or at least four chemotherapy agents). In some embodiments themethod further comprises administering to the individual an effectiveamount of a therapeutic antibody (e.g., at least one therapeuticantibody, such as at least two, at least three, or at least fourtherapeutic antibodies). Additionally or alternatively, in someembodiments the method further comprises administering to the individualan effective amount of an immunotherapeutic agent (e.g., at least oneimmunotherapeutic agent, such as at least two, at least three, or atleast four immunotherapeutic agents). Additionally or alternatively, insome embodiments, the method comprises administering the polypeptide andthe chemotherapy agent in combination with one or more additional modesof therapy, including, but not limited to, e.g., radiation therapy,surgery, cryoablation, and bone marrow transplant.

In some embodiments, the agent that blocks the interaction between CD47(e.g., hCD47) and SIRPα (e.g., hSIRPα) is a small molecule inhibitor ofthe CD47-SIRPα pathway (e.g., RRX-001 and others). See, e.g., Miller etal. (2019) “Quantitative high-throughput screening assays for thediscovery and development of SIRPα-CD47 interaction inhibitors.” PLoSONE 14(7): e0218897 and Sasikumar et al. ACR-NCI-EORTC InternationalConference: Molecular Targets and Cancer Therapeutics; Oct. 26-30, 2017;Philadelphia, Pa.; Abstract B007.

In some embodiments, the agent that blocks the interaction between CD47(e.g., hCD47) and SIRPα (e.g., hSIRPα) binds CD47 (e.g., hCD47). In someembodiments, the agent binds CD47 (e.g., hCD47) with a K_(D) of about 10nM or better (such as at least about any one of 9 nM, 8 nM, 7 nM, 6 nM,5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 200 pM, 100 pM, 50 pM,25 pM, 20 pM 10 pM or less than 10 pM). In some embodiments, the agentthat binds CD47 (e.g., hCD47) exhibits at least about 50% CD47 receptoroccupancy (e.g., at least about any one of 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 99%, or about 100%) in a human subject. In someembodiments, the agent that binds CD47 (e.g., hCD47) has an EC50 ofabout 80 ng/ml or less, e.g., about any one of 75, 70, 65, 60, 55, 50,45, 40, 35, 30, 25, 20, 15, 10, or 5 ng/ml. In some embodiments, theagent that binds CD47 (e.g., hCD47) is an anti-CD47 antibody (e.g., atherapeutic anti-CD47 antibody) or an antigen-binding fragment thereof.In some embodiments, the antigen binding fragment is a Fab, a Fab′, aFab′-SH, an F(ab′)2, an Fv, an scFv, a one-armed antibody, or a diabody.In some embodiments, the anti-CD47 antibody is a monospecific antibody.In some embodiments, the anti-CD47 antibody is a multispecific (e.g.,bispecific) antibody. In some embodiments the term “anti-CD47 antibody”encompasses antibody-based constructs (such as multispecific constructs)including, without limitation triomabs, DARTs (i.e., dual-affinityre-targeting antibodies), TandAbs (i.e., tandem diabodies), tandemscFvs, CrossMabs, DNLs (i.e., dock and lock antibodies), DVD-Ig (i.e.,dual variable domain immunoglobulins), tetravalent bispecific IgGs,nanobodies, dual targeting domains, and ART-Igs (i.e., asymmetricreengineering technology-immunoglobulins). Additional details regardingexemplary antibody constructs (both monospecific and multispecific) areprovided in Husain et al. (2018) Biodrugs 32(5): 441-464 and Spiess etal. (2015) Molecular Immunology 67(2): 95-106. In some embodiments, theanti-CD47 antibody is Hu5F9-G4, B6H12.2, BRIC126, CC-90002, SRF231, orIBI188 (from Innovent Biologics) (see, e.g., Zhao et al. (2011), PNASUSA 108:18342-18347; Chao et al. (2010) Cell 142:699-713, Kim et al.(2012) Leukemia 26:2538-2545; Chao et al. (2011) Blood 118:4890-4891;Goto et al. (2014) Eur J. Cancer 50:1836-1846; and Edris et al. (2012)PNAS USA 109:6656-61 for additional information about these anti-CD47antibodies).

In some embodiments, the agent that blocks the interaction between CD47(e.g., hCD47) and SIRPα (e.g., hSIRPα) binds SIRPα (e.g., hSIRPα). Insome embodiments, the agent binds SIRPα (e.g., hSIRPα) with a K_(D) ofabout 10 nM or better (such as at least about any one of 9 nM, 8 nM, 7nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 200 pM, 100pM, 50 pM, 25 pM, 20 pM 10 pM or less than 10 pM). In some embodiments,the agent that binds SIRPα (e.g., hSIRPα) exhibits at least about 50%SIRPα receptor occupancy (e.g., at least about any one of 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%) in a humansubject. In some embodiments, the agent that binds SIRPα (e.g., hSIRPα)has an EC50 of about 80 ng/ml or less, e.g., about any one of 75, 70,65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 ng/ml. In someembodiments, the agent that binds SIRPα (e.g., hSIRPα) is an anti-SIRPαantibody (e.g., a therapeutic anti-SIRPα antibody) or an antigen-bindingfragment thereof. In some embodiments, the antigen binding fragment is aFab, a Fab′, a Fab′-SH, an F(ab′)2, an Fv, an scFv, a one-armedantibody, or a diabody. In some embodiments, the anti-SIRPα antibody isa monospecific antibody or monospecific antibody construct (including,but not limited to those described above). In some embodiments, theanti-SIRPα antibody is a multispecific (e.g., bispecific) antibody or amultispecific antibody construct (including, but not limited to thosedescribed above). In some embodiments, the anti-SIRPα antibody isKWAR23, SE12C3, 040, or MY-1 (see, e.g., Ring et al. (2017) PNAS USA114(49): E10578-E10585); Murata et al. (2018) Cancer Sci109(5):1300-1308; and Yanigata et al. (2017) JCI Insight 2:e89140 foradditional information about these anti-SIRPα antibodies). In someembodiments, the anti-SIRPα antibody is an antibody described in WO2018/057669; US-2018-0105600-A1; US20180312587; WO2018107058;WO2019023347; US20180037652; WO2018210795; WO2017178653; WO2018149938;WO2017068164; and WO2016063233, the contents of which are incorporatedherein by reference in their entireties.

In some embodiments, the agent that blocks the interaction between CD47(e.g., hCD47) and SIRPα (e.g., hSIRPα) is an anti-SIRPβ antibody or ananti-SIRPγ antibody (e.g., an anti-SIRPβ antibody or anti-SIRPγ antibodythat is capable of binding SIRPα), or an antigen-binding fragmentthereof. In some embodiments, the agent is an antibody (or antigenbinding fragment thereof) that is capable of bind two or more of SIRPα,SIRPβ, and SIRPγ. In some embodiments, such antibody binds SIRPα (e.g.,hSIRPα) with a K_(D) of about 10 nM or better (such as at least aboutany one of 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500pM, 250 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 10 pM or less than 10pM). In some embodiments, the antibody exhibits at least about 50% SIRPαreceptor occupancy (e.g., at least about any one of 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%) in a human subject. Insome embodiments, the antibody has an EC50 of about 80 ng/ml or less,e.g., about any one of 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20,15, 10, or 5 ng/ml. In some embodiments, the antigen binding fragment isa Fab, a Fab′, a Fab′-SH, an F(ab′)2, an Fv, an scFv, a one-armedantibody, or a diabody. In some embodiments, the antibody is amonospecific antibody or monospecific antibody construct (including, butnot limited to those described above). In some embodiments, the antibodyis a multispecific (e.g., bispecific) antibody or a multispecificantibody construct (including, but not limited to those describedabove).

In some embodiments, the agent that blocks the interaction between CD47(e.g., hCD47) and SIRPα (e.g., hSIRPα) is a fusion polypeptidecomprising a moiety that binds CD47. In some embodiments, the fusionpolypeptide comprises an antibody Fc region and a moiety that bindsCD47. In some embodiments, the portion of the fusion polypeptide thatbinds CD47 (e.g., hCD47) binds CD47 (e.g., hCD47) with a K_(D) of about10 nM or better (such as at least about any one of 9 nM, 8 nM, 7 nM, 6nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 20 OpM, 100 pM, 50pM, 25 pM, 20 pM, 10 pM or less than 10 pM). In some embodiments, thefusion polypeptide exhibits at least about 50% CD47 receptor occupancy(e.g., at least about any one of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 99%, or about 100%) in a human subject. In some embodiments,the fusion polypeptide has an EC50 of about 80 ng/ml or less, e.g.,about any one of 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10,or 5 ng/ml. In some embodiments, the fusion polypeptide comprises WThuman antibody Fc region. In some embodiments, the fusion polypeptidecomprises an Fc variant (e.g., a variant of a WT human antibody Fcregion) that exhibits reduced (e.g., such as ablated) effector functionas compared to a WT Fc region. Exemplary Fc variants are described in WO2017/027422 and US 2017/0107270, the contents of which are incorporatedherein by reference in their entireties. In some embodiments, moietythat binds CD47 (e.g., hCD47) is a WT SIRPα (e.g., hSIRPα), or a WTSIRPγ (e.g., hSIRPγ). In some embodiments, moiety that binds CD47 (e.g.,hCD47) is a CD47-binding fragment (e.g., d1 domain) of a WT SIRPα (e.g.,hSIRPα), or a WT SIRPγ (e.g., hSIRPγ). In some embodiments, the moietythat binds CD47 (e.g., hCD47) is a SIRPα variant, a SIRPγ variant, aSIRPβ variant, or a CD47-binding fragment thereof (e.g., the d1 domain).Exemplary SIRPγ variants, SIRPβ1 variant, and SIRPβ2 variants aredescribed in, e.g., WO 2013/109752; US 2015/0071905; U.S. Pat. No.9,944,911; WO 2016/023040; WO 2017/027422; US 2017/0107270; U.S. Pat.Nos. 10,259,859; 9,845,345; WO2016187226; US20180155405; WO2017177333;WO2014094122; US2015329616; US20180312563; WO2018176132; WO2018081898;WO2018081897; PCT/US2019/048921; US20180141986A1; and EP3287470A1, thecontents of which are incorporated herein by reference in theirentireties.

In some embodiments, the agent that blocks the interaction between CD47(e.g., hCD47) and SIRPα (e.g., hSIRPα) is a fusion polypeptidecomprising an antibody Fc region and a SIRPα variant. In someembodiments, the SIRPα variant binds CD47 (e.g., hCD47) with a K_(D) ofabout 10 nM or better (such as at least about any one of 9 nM, 8 nM, 7nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 20 OpM, 100pM, 50 pM, 25 pM, 20 pM, 10 pM or less than 10 pM). In some embodiments,the fusion polypeptide exhibits at least about 50% CD47 receptoroccupancy (e.g., at least about any one of 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 99%, or about 100%) in a human subject. In someembodiments, the fusion polypeptide has an EC50 of about 80 ng/ml orless, e.g., about any one of 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25,20, 15, 10, or 5 ng/ml. In some embodiments, the fusion polypeptidecomprises WT human antibody Fc region. In some embodiments, the fusionpolypeptide comprises an Fc variant (e.g., a variant of a WT humanantibody Fc region) that exhibits reduced (e.g., such as ablated)effector function as compared to a WT Fc region, such as those describedin the references cited herein. In some embodiments, the fusionpolypeptide comprises a SIRPα variant described in WO 2013/109752; US2015/0071905; WO 2016/023040; WO 2017/027422; US 2017/0107270; U.S. Pat.Nos. 10,259,859; 9,845,345; WO2016187226; US20180155405; WO2017177333;WO2014094122; US2015329616; US20180312563; WO2018176132; WO2018081898;WO2018081897; US20180141986A1; and EP3287470A1, the contents of whichare incorporated herein by reference in their entireties. In someembodiments, the fusion polypeptide comprising an antibody Fc region anda SIRPα variant is TTI-621, TTI-622, or IMM01 (see, e.g., Petrova et al.(2017) Clin Cancer Res 23:1086-1079; Russ et al. (2018) Blood Rev50268-960X(17)30093-0; Zhang, X, Chen, W, Fan, J et al. DisruptingCD47-SIRPα axis alone or combined with autophagy depletion for thetherapy of glioblastoma. Carcinogenesis 2018; 39: 689-99).

In some embodiments, the agent that blocks the interaction between CD47(e.g., hCD47) and SIRPα (e.g., hSIRPα) is a fusion polypeptidecomprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variantdescribed herein) and an Fc domain variant (e.g., an Fc domain variantdescribed herein).

In some embodiments, provided is a method of treating cancer (e.g.,leukemia, such as acute lymphoblastic leukemia) in an individual (e.g.,a human individual), comprising administering to the individual aneffective amount of (a) an agent that blocks the interaction betweenCD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) and (b) an BCL2 inhibitor(e.g., a selective BCL2 inhibitor, such as venetoclax). In someembodiments, the agent is a polypeptide comprising a SIRPα D1 domainvariant and an Fc domain variant) wherein the SIRPα D1 domain variantcomprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85,wherein the Fc domain variant is (i) a human IgG1 Fc region comprisingL234A, L235A, G237A, and N297A mutations, wherein numbering is accordingto the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S,P331S, and N297A mutations, wherein numbering is according to the EUindex of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P,F234V, L235A, and delG236 mutations, wherein numbering is according tothe EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P,E233P, F234V, L235A, delG236, and N297A mutations, wherein numbering isaccording to the EU index of Kabat.

In some embodiments, provided is a method of treating cancer (e.g.,colon cancer) in an individual (e.g., a human individual), comprisingadministering to the individual an effective amount of (a) an agent thatblocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g.,hSIRPα), and (b) platinum-based chemotherapy agent (e.g., cisplatin). Insome embodiments, the agent is a polypeptide comprising a SIRPα D1domain variant and an Fc domain variant, wherein the SIRPα D1 domainvariant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat.

In some embodiments, provided is a method of treating cancer (e.g., headand neck cancer, such as head and neck squamous cell carcinoma) in anindividual (e.g., a human individual), comprising administering to theindividual an effective amount of (a) an agent that blocks theinteraction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) (b) aPD-1 inhibitor, (c) an anti-metabolite, and (d) a platinum-basedchemotherapy agent. In some embodiments, the agent that blocks theinteraction between CD47 and SIRPα is a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant) wherein the SIRPα D1 domainvariant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO:85, wherein the Fc domain variant is (i) a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat.

In some embodiments, provided is a method of treating cancer (e.g.,gastric cancer or gastroesophageal cancer) in an individual (e.g., ahuman individual), comprising administering to the individual aneffective amount of (a) an agent that blocks the interaction betweenCD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) (b) an anti-HER2 antibody,(c) an anti-VEGFR2 antibody, and (d) paclitaxel. In some embodiments,the agent that blocks the interaction between CD47 and SIRPα is apolypeptide comprising a SIRPα D1 domain variant and an Fc domainvariant) wherein the SIRPα D1 domain variant comprises the amino acidsequence of SEQ ID NO: 81 or SEQ ID NO: 85, wherein the Fc domainvariant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A,and N297A mutations, wherein numbering is according to the EU index ofKabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297Amutations, wherein numbering is according to the EU index of Kabat;(iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, anddelG236 mutations, wherein numbering is according to the EU index ofKabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V,L235A, delG236, and N297A mutations, wherein numbering is according tothe EU index of Kabat.

Further details regarding the methods of treatment with polypeptidescomprising a SIRPα D1 domain variant and an Fc domain variant aredescribed below. See also WO 2017/027422 and U.S. Pat. No. 10,259,859,the contents of each of which are incorporated by reference herein intheir entireties.

Signal-Regulatory Protein α (SIRPα) D1 Domain and Variants Thereof

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein a (SIRP-α) D1 variant comprising a SIRPα D1domain, or a fragment thereof, that comprises an amino acid mutation atresidue 80 relative to a wild-type SIRPα D1 domain (e.g., a wild-typeSIRPα D1 domain set forth in SEQ ID NO: 1 or 2); and at least oneadditional amino acid mutation relative to a wild-type SIRPα D1 domain(e.g., a wild-type SIRPα D1 domain set forth in SEQ ID NO: 1 or 2) at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92.

Also disclosed herein, in some embodiments, are polypeptides comprisingan Fc domain variants, wherein an Fc domain variant dimer comprises twoFc domain variants, wherein each Fc domain variant independently isselected from (i) a human IgG1 Fc region consisting of mutations L234A,L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting ofmutations A330S, P331S and N297A; or (iii) a human IgG4 Fc regioncomprising mutations S228P, E233P, F234V, L235A, delG236, and N297A.

Signal-regulatory protein α (“SIRP-α” or “SIRP-alpha”) is atransmembrane glycoprotein belonging to the Ig superfamily that iswidely expressed on the membrane of myeloid cells. SIRPα interacts withCD47, a protein broadly expressed on many cell types in the body. Theinteraction of SIRPα with CD47 prevents engulfment of “self” cells,which can otherwise be recognized by the immune system. It has beenobserved that high CD47 expression on tumor cells can act, in acutemyeloid leukemia and several solid tumor cancers, as a negativeprognostic factor for survival.

Native SIRPα comprises 3 highly homologous immunoglobulin (Ig)-likeextracellular domains—D1, D2, and D3. The SIRPα D1 domain (“D1 domain”)refers to the membrane distal, extracellular domain of SIRPα andmediates binding of SIRPα to CD47. As used herein, the term “SIRPαpolypeptide” refers to any SIRPα polypeptide or fragment thereof that iscapable of binding to CD47. There are at least ten variants of wild-typehuman SIRPα. Table 1 shows the amino acid sequences of the D1 domains ofthe naturally occurring wild-type human SIRPα D1 domain variants (SEQ IDNOs: 1 and 2). In some embodiments, a SIRPα polypeptide comprises aSIRPα D1 domain. In some embodiments, a SIRPα polypeptide comprises awild-type D1 domain, such as those provided in SEQ ID NOs: 1 and 2. Insome embodiments, a SIRPα polypeptide includes a D2 or D3 domain (orboth a D2 and a D3 domain) (see Table 3) of a wild-type human SIRPα.

TABLE 1 Sequences of Wild-Type SIRPα D1 Domains SEQ ID NO: DescriptionAmino Acid Sequence 1 Wild-type D1 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQdomain variant 1 WFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGA GTELSVRAKPS 2 Wild-type D1EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQ domain variant 2WFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGT ELSVRAKPS 11 Wild-type pan-D1EEX₁LQVIQPDKX₂VX₃VAAGEX₄AX5LX₆CTX₇TSLIP domainVGPIQWFRGAGPX₈RELIYNQKEGHFPRVTTVSX₉X₁₀  Amino acidTKRX₁₁NMDFX₁₂IX₁₃IX₁₄NITPADAGTYYCVKFRKGS substitutionsX₁₅X₁₆DX₁₇EFKSGAGTELSVRX₁₈KPS relative to SEQ IDX₁ is E or G; X₂ is S or F; X₃ is L or S;  NO: 11X₄ is T or S; X₅ is T or I; X₆ is R, H,or L; X₇ is A or V; X₈ is G or A; X₉ is Dor E; X₁₀ is L or S; X₁₁ is N or E or D; X₁₂ is S or P; X₁₃ is R or S; X₁₄ is G orS; X₁₅ is P or absent; X₁₆ is D or P; X₁₇  is V or T; and X₁₈ is A or G

As used herein, the term “SIRPα D1 domain variant” refers to apolypeptide comprising a SIRPα D1 domain or a CD47-binding portion of aSIRPα polypeptide that has a higher affinity to CD47 than wild-typeSIRPα. A SIRPα D1 domain variant comprises at least one amino acidsubstitution, deletion, or insertion (or a combination thereof) relativeto a wild-type SIRPα.

In some embodiments, SIRPα D1 domain variants disclosed herein comprisea SIRPα D1 domain or variant thereof. In some embodiments, a SIRPα D1domain variant comprises one or more amino acid substitutions,insertions, additions, or deletions relative to a wild-type D1 domainshown in SEQ ID NOs: 1 and 2. Table 2 lists exemplary amino acidsubstitutions in each SIRPα D1 domain variant (SEQ ID NOs: 13-14). Insome embodiments, the SIRPα D1 domain polypeptide or SIRPα D1 domainvariant comprises a fragment of the D1 domain. In some embodiments, theSIRPα polypeptide fragment or SIRPα D1 domain variant fragment comprisesan amino acid sequence of less than 10 amino acids in length, about 10amino acids in length, about 20 amino acids in length, about 30 aminoacids in length, about 40 amino acids in length, about 50 amino acids inlength, about 60 amino acids in length, about 70 amino acids in length,about 80 amino acids in length, about 90 amino acids in length, about100 amino acids in length, or more than about 100 amino acids in length.In some embodiments, the SIRPα D1 domain fragments retain the ability tobind to CD47.

In some embodiments, a polypeptide of the disclosure comprising a SIRPαD1 domain variant binds with higher binding affinity to CD47 than awild-type human SIRPα D1 domain. In some embodiments, the SIRPα D1domain variant binds to human CD47 with at least 1-fold (e.g., at least1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold or greaterthan 5-fold) affinity than the affinity of a naturally occurring D1domain. In some embodiments, the SIRPα D1 domain variant binds to humanCD47 with at least 1-fold (e.g., at least 10-fold, 100-fold, 1000-foldor greater than 1000-fold) affinity than the affinity of a naturallyoccurring D1 domain.

As used herein, the term “optimized affinity” or “optimized bindingaffinity” refers to an optimized strength of the binding interactionbetween a polypeptide disclosed herein, including a SIRPα D1 domainvariant, and CD47. For example, in some embodiments, the polypeptidebinds primarily or with higher affinity to CD47 on cancer cells and doesnot substantially bind or binds with lower affinity to CD47 onnon-cancer cells. In some embodiments, the binding affinity between thepolypeptide and CD47 is optimized such that the interaction does notcause clinically relevant toxicity or decreases toxicity compared to avariant which binds with maximal affinity. In some embodiments, in orderto achieve an optimized binding affinity between a polypeptide providedherein and CD47, the polypeptide including a SIRPα D1 domain variant isdeveloped to have a lower binding affinity to CD47 than which ismaximally achievable. In some embodiments, the SIRPα D1 domain variantsdisclosed herein cross react with rodent, non-human primate (NHP), andhuman CD47.

As used herein, the term “immunogenicity” refers to the property of aprotein (e.g., a therapeutic protein) which causes an immune response inthe host as though it is a foreign antigen. The immunogenicity of aprotein can be assayed in vitro in a variety of different ways, such asthrough in vitro T-cell proliferation assays.

As used herein, the term “minimal immunogenicity” refers to animmunogenicity of a protein (e.g., a therapeutic protein) that has beenmodified, e.g., through amino acid substitutions, to be lower (e.g., atleast 10%, 25%, 50%, or 100% lower) than the immunogenicity before theamino acid substitutions are introduced (e.g., an unmodified protein).In some embodiments, a protein (e.g., a therapeutic protein) is modifiedto have minimal immunogenicity and causes no or very little host immuneresponse even though it is a foreign antigen.

In some embodiments, the SIRPα D1 domain variant demonstrates minimalimmunogenicity. In some embodiments, a SIRPα polypeptide of thedisclosure administered to a subject has the same amino acid sequence asthat of the SIRPα polypeptide in a biological sample of the subject,except for amino acid changes which increase affinity of the SIRPα D1domain variant. In some embodiments, the polypeptide variants disclosedherein lower the risk of side effects compared to anti-CD47 antibodiesor wild-type SIRPα. In some embodiments, the polypeptide variantsdisclosed herein lower the risk of anemia compared to anti-CD47antibodies or wild-type SIRPα. In some embodiments, the polypeptidevariants disclosed herein do not cause acute anemia in rodent ornon-human primates (NHP) studies.

Table 2 lists specific amino acid substitutions in a SIRPα D1 domainvariant relative to each D1 domain sequence. In some embodiments, aSIRPα D1 domain variant includes one or more (e.g., two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteenor more) of the substitutions listed in Table 2. In some embodiments, aSIRPα D1 domain variant includes at most fourteen amino acidsubstitutions relative to a wild-type D1 domain. In some embodiments, aSIRPα D1 domain variant includes at most ten amino acid substitutionsrelative to a wild-type D1 domain. In some embodiments, a SIRPα D1domain variant includes at most seven amino acid substitutions relativeto a wild-type D1 domain. In some embodiments, a SIRPα D1 domain variantof the disclosure has at least 90% (e.g., at least 92%, 95%, 97% orgreater than 97%) amino acid sequence identity to a sequence of awild-type D1 domain.

In some embodiments, a SIRPα D1 domain variant is a chimeric SIRPα D1domain variant that includes a portion of two or more wild-type D1domains or variants thereof (e.g., a portion of one wild-type D1 domainor variant thereof and a portion of another wild-type D1 domain orvariant thereof). In some embodiments, a chimeric SIRPα D1 domainvariant includes at least two portions (e.g., three, four, five or moreportions) of wild-type D1 domains or variants thereof, wherein each ofthe portions is from a different wild-type D1 domain. In someembodiments, a chimeric SIRPα D1 domain variant further includes one ormore amino acid substitutions listed in Table 2.

TABLE 2 Amino Acid Substitutions in a SIRPα D1 Domain Variant SEQ ID NO:Description Amino Acid Sequence 13 D1 domain v1EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGNITPADAGTYYCX₁₂KX₁₃RKGSPDD VEX₁₄KSGAGTELSVRAKPS —Amino acid X₁ = L, I, V; X₂ = V, L, I; X₃ = A, V;  substitutionsX₄ = A, I, L; X₅ = I, T, S, F; X₆ = E, relative to SEQ IDV, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, NO: 13R; X₁₀ = L, T, G; X₁₁ = K, R; X₁₂ = V, I; X₁₃ = F, L, V; X₁₄ = F, V 14D1 domain v2 EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISNITPADAGTYYCX₁₂KX₁₃RKGSPDTEX₁₄ KSGAGTELSVRAKPS — Amino acidX₁= L, I, V; X₂ = V, L, I; X₃ = A, V;  substitutionsX₄ = V, I, L; X₅ = I, T, S, F; X₆ = E, V, relative to SEQ IDL; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; NO: 14X₁₀ = S, T, G; X₁₁ = K, R; X₁₂ = V, I; X₁₃ = F, L, V; X₁₄ = F, V 23Pan D1 domain EEX₁X₂QX₃IQPDKX₄VX₅VAAGEX₆X₇X₈LX₉CTX₁₀TSLX₁₁PVGPIQWFRGAGPX₁₂RX₁₃LIYNQX₁₄X₁₅GX₁₆FPRVTTVSX₁₇X₁₈TX₁₉RX₂₀NMDFX₂₁IX₂₂IX₂₃NITPADAGTYYCX₂₄KX₂₅RKGSPDX₂₆X₂₇EX₂₈KSGAGTELSVRX₂₉KPS — Amino acidX₁ = E, G; X₂ = L, I, V; X₃ = V, L, I; substitutionsX₄ = S, F; X₅ = L, S; X₆ = S, T; X₇ = A, relative to SEQ IDV; X₈ = I, T; X₉ = H, R; X₁₀ = A, V, I, L;  NO: 23X₁₁ = I, T, S, F; X₁₂ = A, G; X₁₃ = E, V, L;X₁₄ = K, R; X₁₅ = E, Q; X₁₆ = H, P, R; X₁₇ =D, E; X₁₈ = S, L, T, G; X₁₉ = K, R; X₂₀ = E, D; X₂₁ = S, P; X₂₂ = S, R; X₂₃ = S, G; X₂₄ =V, I; X₂₅ = F, L, V; X₂₆ = D or absent;X₂₇ = T, V; X₂₈ = F, V; and X₂₉ = A, G

In some embodiments, a polypeptide comprises a SIRPα D1 domain variantthat comprises a sequence of:EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGNITPADAGTYYCX₁₂KX₁₃RKGSPDDVEX₁₄KSGAGTELSVRAKPS (SEQ ID NO: 13), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is L, T, or G; X₁₁ is Kor R; X₁₂ is V or I; X₁₃ is F, L, or V; and X₁₄ is F or V; and whereinthe variant comprises at least one amino acid substitution relative to awild-type SIRPα D1 domain that comprises the sequence of SEQ ID NO: 1.

In some embodiments, a polypeptide comprises a SIRPα D1 domain variantthat comprises the sequence of SEQ ID NOs: 13, wherein X₁ is L, I, or V.In any of the aforementioned embodiments, X₂ is V, L, or, I. In someembodiments, X₃ is A or V. In some embodiments, X₄ is A, I, or L. Insome embodiments, X₅ is I, T, S, or F. In some embodiments, X₆ is E, V,or L. In some embodiments, X₇ is K or R. In some embodiments, X₈ is E orQ. In some embodiments, X₉ is H, P, or R. In some embodiments, X₁₀ is L,T, or G. In some embodiments, X₁₁ is K or R. In some embodiments, X₁₂ isV or I. In some embodiments, X₁₃ is F, L, V. In some embodiments, X₁₄ isF or V. In some embodiments, the polypeptide of this aspect of thedisclosure includes no more than six amino acid substitutions relativeto the wild-type SIRPα D1 domain that comprises the sequence of SEQ IDNO: 1.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain thatcomprises the sequence of SEQ ID NO: 1. In some embodiments, thepolypeptide binds CD47 with at least 100-fold greater binding affinitythan the wild-type SIRPα D1 domain that comprises the sequence of SEQ IDNO: 1. In some embodiments, the polypeptide binds CD47 with at least1000-fold greater binding affinity than the wild-type SIRPα D1 domainthat comprises the sequence of SEQ ID NO: 1. In some embodiments, aSIRPα D1 domain variant polypeptide or fragment thereof binds to CD47with a K_(D) less than 1×10⁻⁸M, less than 5×10⁻⁹M, less than 1×10⁻⁹M,less 5×10⁻¹⁰ M, less than 1×10⁻¹⁰ M or less than 1×10⁻¹¹M. In someembodiments, a SIRPα D1 domain variant polypeptide or fragment thereofbinds to CD47 with a K_(D) between about 500 nM and 100 nM, betweenabout 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM,between about 500 pM and 100 pM, between about 100 pM and 50 pM, orbetween about 50 pM and 10 pM.

In some embodiments, a polypeptide includes a SIRPα D1 domain variantthat comprises a sequence of:EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISNITPADAGTYYCX₁₂KX₁₃RKGSPDTEXHKSGAGTELSVR AKPS(SEQ ID NO: 14), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃ is A orV; X₄ is V, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇ is K orR; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is S, T, or G; X₁₁ is K or R; X₁₂is V or I; X₁₃ is F, L, or V; and X₁₄ is F or V; and wherein the variantcomprises at least one amino acid substitution relative to a wild-typeSIRPα D1 domain that comprises the sequence of SEQ ID NO: 2.

In some embodiments in this aspect of the disclosure, the polypeptidecomprises the sequence of SEQ ID NO: 14, wherein X₁ is L, I, or V. Insome embodiments, X₂ is V, L, or, I. In some embodiments, X₃ is A or V.In some embodiments, X₄ is V, I, or L. In some embodiments, X₅ is I, T,S, or F. In some embodiments, X₆ is E, V, or L. In some embodiments, X₇is K or R. In some embodiments, X₈ is E or Q. In some embodiments, X₉ isH, P, or R. In some embodiments, X₁₀ is S, T, or G. In some embodiments,X₁₁ is K or R. In some embodiments, X₁₂ is V or I. In some embodiments,X₁₃ is F, L, or V. In some embodiments, X₁₄ is F or V. In someembodiments, the polypeptide of this aspect of the disclosure includesno more than six amino acid substitutions relative to the wild-typeSIRPα D1 domain that comprises the sequence of SEQ ID NO: 2.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of SEQ ID NO: 2. In some embodiments, the polypeptide bindsCD47 with at least 100-fold greater binding affinity than the wild-typeSIRPα D1 domain having the sequence of SEQ ID NO: 2. In someembodiments, the polypeptide binds CD47 with at least 1000-fold greaterbinding affinity than the wild-type SIRPα D1 domain having the sequenceof SEQ ID NO: 2. In some embodiments, a SIRPα D1 domain variantpolypeptide or fragment thereof binds to CD47 with a K_(D) less than1×10⁻⁸M, less than 5×10⁻⁹M, less than 1×10⁻⁹M, less 5×10⁻¹⁹M, less than1×10⁻¹⁹M or less than 1×10⁻¹¹M. In some embodiments, a SIRPα D1 domainvariant polypeptide or fragment thereof binds to CD47 with a K_(D)between about 500 nM and 100 nM, between about 100 nM and 50 nM, betweenabout 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nMand 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM.

In some embodiments, a polypeptide includes a SIRPα D1 domain varianthaving a sequence of:EEX₁X₂QX₃IQPDKX₄VX₅VAAGEX₆X₇X₈LX₉CTX₁₀TSLX₁₁PVGPIQWFRGAGPX₁₂RX₁₃LIYNQX₁₄X₁₅GX₁₆FPRVTTVSX₁₇X₁₈TX₁₉RX₂₀NMDFX₂₁IX₂₂IX₂₃NITPADAGTYYCX₂₄KX₂₅RKGSPDX₂₆X₂₇EX₂₈KSGAGTELSVRX₂₉KPS(SEQ ID NO: 23), wherein X₁ is E or G; X₂ is L, I, or V; X₃ is V, L, or,I; X₄ is S or F; X₅ is L or S; X₆ is S or T; X₇ is A or V; X₈ is I or T;X₉ is H or R; X₁₀ is A, V, I, or L; X₁₁ is I, T, S, or F; X₁₂ is A or G;X₁₃ is E, V, or L; X₁₄ is K or R; X₁₅ is E or Q; X₁₆ is H, P, or R; X₁₇is D or E; X₁₈ is S, L, T, or G; X₁₉ is K or R; X₂₀ is E or D; X₂₁ is Sor P; X₂₂ is S or R; X₂₃ is S or G; X₂₄ is V or I; X₂₅ is F, L, V; X₂₆is D or absent; X₂₇ is T or V; X₂₈ is F or V; and X₂₉ is A or G; andwherein the variant comprises at least one amino acid substitutionrelative to a wild-type SIRPα D1 domain having the sequence of SEQ IDNO: 1 or 2.

In any of the aforementioned embodiments in this aspect of thedisclosure, X₂ is L, I, or V. In any of the aforementioned embodiments,X₃ is V, L, or, I. In embodiments, X₄ is S or F. In some embodiments, X₅is L or S. In some embodiments, X₆ is S or T. In some embodiments, X₇ isA or V. In some embodiments, X₈ is I or T. In some embodiments, X₉ is Hor R. In some embodiments, X₁₀ is A, V, I, or L. In some embodiments,X₁₁ is I, T, S, or F. In some embodiments, X₁₂ is A or G. In someembodiments, X₁₃ is E, V, or L. In some embodiments, X₁₄ is K or R. Insome embodiments, X₁₅ is E or Q. In some embodiments, X₁₆ is H, P, or R.In some embodiments, X₁₇ is D or E. In some embodiments, X₁₈ is S, L, T,or G. In some embodiments, X₁₉ is K or R. In some embodiments, X₂₀ is Eor D. In some embodiments, X₂₁ is S or P. In some embodiments, X₂₂ is Sor R. In some embodiments, X₂₃ is S or G. In some embodiments, X₂₄ is Vor I. In some embodiments, X₂₅ is F, L, V. In some embodiments, X₂₆ is Dor absent. In some embodiments, X₂₇ is T or V. In some embodiments, X₂₈is F or V. In some embodiments, X₂₉ is A or G. In some embodiments, thepolypeptide of this aspect of the disclosure includes no more than sixamino acid substitutions relative to the wild-type SIRPα D1 domainhaving the sequence of SEQ ID NO: 1 or 2.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of SEQ ID NO: 1 or 2. In some embodiments, the polypeptidebinds CD47 with at least 100-fold greater binding affinity than thewild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. Insome embodiments, the polypeptide binds CD47 with at least 1000-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of SEQ ID NO: 1 or 2. In some embodiments, a SIRPα D1 domainvariant polypeptide or fragment thereof binds to CD47 with a K_(D) lessthan 1×10⁻⁸M, less than 5×10⁻⁹M, less than 1×10⁻⁹M, less 5×10⁻¹⁰ M, lessthan 1×10⁻¹⁰ M or less than 1×10⁻¹¹M. In some embodiments, a SIRPα D1domain variant polypeptide or fragment thereof binds to CD47 with aK_(D) between about 500 nM and 100 nM, between about 100 nM and 50 nM,between about 50 nM and 10 nM, between about 10 nM and 5 nM, betweenabout 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pMand 100 pM, between about 100 pM and 50 pM, or between about 50 pM and10 pM.

In some embodiments, a polypeptide of the disclosure including a SIRPαD1 domain variant further comprises a D2 domain having the sequence ofSEQ ID NO: 24, a D3 domain having the sequence of SEQ ID NO: 25, or a D2domain having the sequence of SEQ ID NO: 24 and a D3 domain having thesequence of SEQ ID NO: 25 of a wild-type human SIRPα as shown in Table3. In some embodiments, the SIRPα D1 domain variant further comprises afragment or variant of a D2 domain or a fragment or variant of a D3domain. In some embodiments, the SIRPα D1 domain variant furthercomprises a fragment or variant of a D2 domain and a fragment or variantof a D3 domain. In some embodiments, a SIRPα D1 domain variant is joinedto a D2 or D3 domain by way of a linker. In some embodiments, a SIRPα D1domain variant is joined to a D2 and D3 domain by way of a linker.

TABLE 3 Amino Acid Sequences of SIRPα D2 and D3 Domains SEQ ID NO:Description Amino Acid Sequence 24 SIRPα D2APVVSGPAARATPQHTVSFTCESHGFSPRDITL domainKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKV VLTREDVHSQVICEVAHVTLQGDPLRGTANLSE TIR25 SIRPα D3 VPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQ domainLTWLENGNVSRTETASTVTENKDGTYNWMSWLL VNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVS

In some embodiments, a polypeptide of the disclosure including a SIRPαD1 domain variant is attached to an Fc domain variant in order toimprove the pharmacokinetic properties of the polypeptide, e.g.,increase serum half-life. In some embodiments, a SIRPα D1 domain variantis attached to an Fc domain variant that is unable to dimerize. In someembodiments, Fc domain variants serve to increase the serum half-life ofthe polypeptides described herein. In some embodiments, a polypeptide ofthe disclosure including a SIRPα D1 domain variant does not include thesequence of any one of SEQ ID NOs: 26-36 shown in Table 4.

TABLE 4 SEQ ID NO: AMINO ACID SEQUENCE 26EEELQVIQPDKSVSVAAGESAILHCTITSLIPVGPIQWFRGAGPARELIYNQREGHFPRVTTVSETTRRENMDFSISISNITPADAGTYYCVKFRKGSPDTEVKSGAGTELSVRAKPS 27EEEVQVIQPDKSVSVAAGESAILHCTLTSLIPVGPIQWFRGAGPARVLIYNQRQGHFPRVTTVSEGTRRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAGTELSVRAKPS 28EEEVQIIQPDKSVSVAAGESVILHCTITSLTPVGPIQWFRGAGPARLLIYNQREGPFPRVTTVSETTRRENMDFSISISNITPADAGTYYCVKLRKGSPDTEFKSGAGTELSVRAKPS 29EEELQIIQPDKSVSVAAGESAILHCTITSLSPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSEGTKRENMDFSISISNITPADAGTYYCIKLRKGSPDTEFKSGAGTELSVRAKPS 30EEEIQVIQPDKSVSVAAGESVIIHCTVTSLFPVGPIQWFRGAGPARVLIYNQRQGRFPRVTTVSEGTKRENMDFSISISNITPADAGTYYCVKVRKGSPDTEVKSGAGTELSVRAKPS 31EEEVQIIQPDKSVSVAAGESIILHCTVTSLFPVGPIQWFRGAGPARVLIYNQREGRFPRVTTVSEGTRRENMDFSISISNITPADAGTYYCIKLRKGSPDTEFKSGAGTELSVRAKPS 32EEEVQLIQPDKSVSVAAGESAILHCTVTSLFPVGPIQWFRGAGPARVLIYNQREGPFPRVTTVSEGTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEVKSGAGTELSVRAKPS 33EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPS 34EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARLLIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS 35EEEVQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQKQGPFPRVTTISETTRRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAGTELSVRAKPS 36EEELQIIQPDKSVSVAAGESAILHCTITSLTPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSEGTRRENMDFSISISNITPADAGTYYCIKFRKGSPDTEVKSGAGTELSVRAKPS

In some embodiments, the polypeptides and polypeptide constructsdescribed herein are utilized in vitro for binding assays, such asimmune assays. For example, in some embodiments, the polypeptides andpolypeptide constructs described herein are utilized in liquid phase orbound to a solid phase carrier. In some embodiments, polypeptidesutilized for immunoassays are detectably labeled in various ways.

In some embodiments, polypeptides and polypeptide constructs describedherein are bound to various carriers and used to detect the presence ofspecific antigen expressing cells. Examples of carriers include glass,polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,natural and modified celluloses, polyacrylamides, agaroses, andmagnetite. The nature of the carrier can be either soluble or insoluble.

Various different labels and methods of labeling are known. Examples oflabels include enzymes, radioisotopes, fluorescent compounds, colloidalmetals, chemiluminescent compounds, and bio-luminescent compounds.Various techniques for binding labels to polypeptides disclosed hereinare available.

In some embodiments, the polypeptides are coupled to low molecularweight haptens. These haptens are then specifically detected by means ofa second reaction. For example, in some embodiments, the hapten biotinis used with avidin or the haptens dinitrophenol, pyridoxal, orfluorescein are detected with specific anti-hapten antibodies (e.g.,anti-dinitrophenol antibodies, anti-pyridoxal antibodies, andanti-fluorescein antibodies respectively).

SIRPα D1 Domain Variants with Altered Glycosylation Patterns

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein a (SIRP-α) D1 variant comprising a SIRPα D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRPα D1 domain (e.g., a wild-type SIRPα D1domain set forth in SEQ ID NO: 1 or 2); and at least one additionalamino acid mutation relative to a wild-type SIRPα D1 domain (e.g., awild-type SIRPα D1 domain set forth in SEQ ID NO: 1 or 2) at a residueselected from the group consisting of: residue 6, residue 27, residue31, residue 47, residue 53, residue 54, residue 56, residue 66, andresidue 92.

Also disclosed herein, in some embodiments, are polypeptides comprisingan Fc domain variant, wherein an Fc domain variant dimer comprises twoFc domain variants, wherein each Fc domain variant independently isselected from (i) a human IgG1 Fc region consisting of mutations L234A,L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting ofmutations A330S, P331S and N297A; or (iii) a human IgG4 Fc regioncomprising mutations S228P, E233P, F234V, L235A, delG236, and N297A.

In some embodiments, a polypeptide in a composition disclosed hereincomprises a SIRPα D1 domain variant that has reduced or minimalglycosylation. The D1 domain of SEQ ID NOs: 1 and 2 in Table 1 eachcontains a single potential N-linked glycosylation site at amino acidN80 in the sequence N80ITP. Expression of a SIRPα D1 domain in ChineseHamster Ovary (CHO) cells results in a major band of 16 kDa(non-glycosylated) and a minor band of higher molecular weight that wasremoved by Endo Hf. Endo Hf is a recombinant protein fusion ofEndoglycosidase H and maltose binding protein. Endo Hf cleaves withinthe chitobiose core of high mannose and some hybrid oligosaccharidesfrom N-linked glycoproteins. This implies that a proline at amino acidposition 83 can reduce the efficiency of glycosylation, leading to aprotein with different degrees of glycosylation and thereforeheterogeneity. For drug development, heterogeneity can give rise tochallenges in process development. Therefore, to investigate thepossibility of generating homogenous, non-glycosylated forms of SIRPα D1domain variants, in some embodiments, amino acid N80 of a SIRPα D1variant is mutated to Ala. In some embodiments, to make anon-glycosylated, SIRPα D1 domain variant, amino acid N80 in a SIRPα D1domain variant is replaced by any amino acid, including any naturallyand non-naturally occurring amino acid, e.g., N80A and N80Q. In someembodiments, a SIRPα D1 domain variant comprises an N80A mutation and atleast 1 additional mutation (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or10 additional mutations or more). In some embodiments, the additionalmutation is in the CD47 binding site. In some embodiments, theadditional mutation is in the hydrophobic core of the D1 domain.

In some embodiments, a polypeptide in a composition disclosed hereinincludes a SIRPα D1 domain variant that has increased glycosylationrelative to a wild-type SIRPα D1 domain. Another option to increasehomogeneity of the final product is to enhance the efficiency ofglycosylation at amino acid N80 and generate SIRPα D1 domain variantswith increased glycosylation relative to a wild-type. In someembodiments, the amino acid P83 in the sequence NITP83 affects thedegree of glycosylation at amino acid N80. In some embodiments, changingP83 to any amino acid increases the efficiency of glycosylation at N80.In some embodiments, amino acid P83 in a SIRPα D1 domain variant isreplaced by any amino acid, including naturally and non-naturally aminoacids, e.g., P83V, P83A, P831, and P83L. In some embodiments, apolypeptide of the disclosure is expressed in a cell that is optimizednot to glycosylate proteins that are expressed by such cell, for exampleby genetic engineering of the cell line (e.g., genetically engineeredyeast or mammalian host) or modifications of cell culture conditionssuch as addition of kifunensine or by using a naturallynon-glycosylating host such as a prokaryote (E. coli, etc.).

Table 5 lists specific amino acid substitutions in a SIRPα D1 domainvariant relative to each D1 domain variant sequence. In someembodiments, a SIRPα D1 domain variant includes one or more (e.g., two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen or more) of the substitutions listed in Table 5. Insome embodiments, the SIRPα D1 domain variants are not glycosylated orare minimally glycosylated. In some embodiments, the SIRPα D1 domainvariants are fully glycosylated or almost fully glycosylated. In someembodiments, a SIRPα D1 domain variant includes at most fourteen aminoacid substitutions relative to a wild-type D1 domain. In someembodiments, a SIRPα D1 domain variant includes at most ten amino acidsubstitutions relative to a wild-type D1 domain. In some embodiments, aSIRPα D1 domain variant includes at most seven amino acid substitutionsrelative to a wild-type D1 domain. In some embodiments, a SIRPα D1domain variant of the disclosure has at least 90% (e.g., at least 92%,95%, 97% or greater than 97%) amino acid sequence identity to a sequenceof a wild-type D1 domain.

In some embodiments, a SIRPα D1 domain variant is a chimeric SIRPα D1domain variant that includes a portion of two or more wild-type D1domains or variants thereof (e.g., a portion of one wild-type D1 domainor variant thereof and a portion of another wild-type D1 domain orvariant thereof). In some embodiments, a chimeric SIRPα D1 domainvariant includes at least two portions (e.g., three, four, five or moreportions) of wild-type D1 domains or variants thereof, wherein each ofthe portions is from a different wild-type D1 domain. In someembodiments, a chimeric SIRPα D1 domain variant further includes one ormore amino acid substitutions listed in Table 5.

TABLE 5 Amino Acid Substitutions in a SIRPa D1 Domain Variant SEQ ID NO:Description Amino Acid Sequence 37 D1 domain v1EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSP DDVEX₁₆KSGAGTELSVRAKPS —Amino acidX₁ = L, I, V; X₂ = V, L, I; X₃ = A, V; X₄ = A, I, L; X₅ = I, T, S, F;substitutionsX₆ = E, V, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = L, T, G;relative toX₁₁ = K, R; X₁₂ = N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R,SEQ ID NO: 37S, T, V, W, Y; X₁₃ = P, A, C, D, E, F, G, H, I, K, L, M, N, Q,R, S, T, V, W, Y; X₁₄ = V, I; X₁₅ = F, L, V; X₁₆ = F, V 38 D1 domain v2EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDT EX₁₆KSGAGTELSVRAKPS —Amino acidX₁ = L, I, V; X₂ = V, L, I; X₃ = A, V; X₄ = V, I, L; X₅ = I, T, S, F;substitutionsX₆ = E, V, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = S, T, G;relative toX₁₁ = K, R; X₁₂ = N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R,SEQ ID NO: 38S, T, V, W, Y; X₁₃ = P, A, C, D, E, F, G, H, I, K, L, M, N, Q,R, S, T, V, W, Y; X₁₄ = V, I; X₁₅ = F, L, V; X₁₆ = F, V 47 Pan D1 domainEEX₁X₂QX₃IQPDKX₄VX₅VAAGEX₆X₇X₈LX₉CTX₁₀TSLX₁₁PVGPIQWFRGAGPX₁₂RX₁₃LIYNQX₁₄X₁₅GX₁₆FPRVTTVSX₁₇X₁₈TX₁₉RX₂₀NMDFX₂₁IX₂₂IX₂₃X₂₄ITX₂₅ADAGTYYCX₂₆KX₂₇RKGSPDX₂₈X₂₉EX₃₀KSGAGTELSVRX₃₁KP S — Amino acidX₁ = E, G; X₂ = L, I, V; X₃ = V, L, I; X₄ = S, F; X₅ = L, S; X₆ = S,substitutionsT; X₇ = A, V; X₈ = I, T; X₉ = H, R, L; X₁₀ = A, V, I, L; X₁₁ = I, T,relative toS, F; X₁₂ = A, G; X₁₃ = E, V, L; X₁₄ = K, R; X₁₅ = E, Q; X₁₆ = H,SEQ ID NO: 47P, R; X₁₇ = D, E; X₁₈ = S, L, T, G; X₁₉ = K, R; X₂₀ = E, N;A₂₁ = S, P; A₂₂ = S, R; A₂₃ = S, G; A₂₄ = any amino acid;X₂₅ = any amino acid; X₂₆ = V, I; X₂₇ = F, L, V; X₂₈ = D orabsent; X₂₉ = T, V; X₃₀ = F, V; and X₃₁ = A, G 48 Pan D1 domainEEELQX₁IQPDKSVX₂VAAGEX₃AX₄LX₅CTX₆TSLX₇PVGPIQWFRGAGPX₈RX₉LIYNQX₁₀X₁₁GX₁₂FPRVTTVSX₁₃X₁₄TKRX₁₅NMDFSIX₁₆IX₁₇X₁₈ITPADAGTYYCX₁₉KFRKGX₂₀X₂₁X₂₂DX₂₃EFKSGAGTELSVRAKPS — Amino acidX₁ = V, I; X₂ = L, S; X₃ = T, S; X₄ = T, I; X₅ = R, H; X₆ = A, V,substitutionsI; X₇ = I, R, Y, K, F; X₈ = G, A; X₉ = E, V; X₁₀ = K, R; X₁₁ =relative to E, D, Q; X₁₂ = H, P; X₁₃ = D, E; X₁₄ = S, L, T; X₁₅ = N, E;SEQ ID NO: 48X₁₆ = R, S; X₁₇ = G, S; X₁₈ = N, A; X₁₉ = V, I; X₂₀ = S, I, M; X₂₁ = Por absent; X₂₂ = D, P; and X₂₃ = V, T 49 Pan D1 domainEEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDX₈TKRNNMDFSIRIGX₉ITPADAGTYYCX₁₀KFRKGSPDDVEF KSGAGTELSVRAKPS — Amino acidX₁ = V, I, L; X₂ = A, I, V, L; X₃ = I, F, S, T; X₄ = E, V, L; X₅ = K,substitutionsR; X₆ = E, Q; X₇ = H, P, R; X₈ = L, T, S, G; X₉ = A; and X₁₀ = V,relative to I SEQ ID NO: 49 50 Pan D1 domainEEELQX₁IQPDKSVSVAAGESAILHCTX₂TSLX₃PVGPIQWFRGAGPARX₄LIYNQX₅X₆GX₇FPRVTTVSEX₈TKRENMDFSISISX₉ITPADAGTYYCX₁₀KFRKGSPDTEFKS GAGTELSVRAKPS — Amino acidX₁ = V, I; X₂ = V, I; X₃ = I, F; X₄ = E, V; X₅ = K, R; X₆ = E, Q;substitutions X₇ = H, P; X₈ = S, T; X₉ = N, A; and X₁₀ = V, Irelative to SEQ ID NO: 50 51 Pan D1 domainEEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅EGX₆FPRVTTVSDX₇TKRNNMDFSIRIGX₈ITPADAGTYYCX₉KFRKGSPDDVEFKS GAGTELSVRAKPS — Amino acidX₁ = V, I; X₂ = A, I; X₃ = I, F; X₄ = E, V; X₅ = K, R; X₆ = H, P;substitutions X₇ = L, T; X₈ = N, A; and X₉ = V, I relative toSEQ ID NO: 51 52 Pan D1 domain EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRELIYNQX₄EGX₅FPRVTTVSDX₆TKRNNMDFSIRIGX₇ITPADAGTYYCVKFRKGSPDDVEFKSG AGTELSVRAKPS — Amino acidX₁ = V, L, I; X₂ = A, I, L; X₃ = I, T, S, F; X₄ = K, R; X₅ = H, P, R;substitutions X₆ = L, T, G; and X₇ = N, A relative to SEQ ID NO: 52 212 Pan D1 domain EEELQX₁IQPDKSVSVAAGESAILHCTX₂TSLX₃PVGPIQWERGAGPARELIYNQX₄EGX₅FPRVTTVSEX₆TKRENMDFSISISX₇ITPADAGTYYCVKFRKGSPDTEFKSGAG TELSVRAKPS — Amino acidX₁ = V, L, I; X₂ = V, I, L; X₃ = I, T, S, F; X₄ = K, R; X₅ = H, P, R;substitutions L = S, T, G; and X₇ = N, A relative to SEQ ID NO: 212 218 Pan D1 domain EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDX₈TKRNNMDFSIRIGX₉X₁₀X₁₁X₁₂ADAGTYYCX₁₃KFRKGSPD DVEFKSGAGTELSVRAKPS —Amino acidX₁ = V, L, or I; X₂ = A, V, L, or I; X₃ = I, S, T, or F; X₄ = E, L,substitutionsor V; X₅ = K or R; X₆ = E or Q; X₇ = H, R or P; X₈ = S,G, L orrelative to T, X₉ = any amino acid; X₁₀ = any amino acid; X₁₁ = anySEQ ID NO: 218 amino acid; X₁₂ = any amino acid; and X₁₃ = V or I 219 Pan D1 domain EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDX₈TKRNNMDFSIRIGX₉ITX₁₀ADAGTYYCX₁₁KFRKGSPDDVE FKSGAGTELSVRAKPS — Amino acidX₁ = V, L or I; X₂ = A, V, L, or I; X₃ = I, S, T or F; X₄ = E, L, orsubstituionsV; X₅ = K or R; X₆ = E or Q; X₇ = H, R or P; X₈ = S, G, L, or T;relative to X₉ = N; X₁₀ = any amino acid other than P; and X₁₁ = V or ISEQ ID NO: 219

In some embodiments, a polypeptide includes a SIRPα D1 domain varianthaving a sequence of:EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDDVEX₁₆KSGAGTELSVRAKPS (SEQ ID NO: 37), wherein X₁ is L, I, or V; X₂ is V, L, or, I;X₃ is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L;X₇ is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is L, T, or G; X₁₁ isK or R; X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W,or Y; X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, orY; X₁₄ is V or I; X₁₅ is F, L, or V; and X₁₆ is F or V; and wherein thevariant comprises at least one amino acid substitution relative to awild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1.

In some embodiments in this aspect of the disclosure, a polypeptideincludes a SIRPα D1 domain variant having a sequence of SEQ ID NO: 37,wherein X₁ is L, I, or V. In some embodiments, X₂ is V, L, or, I. Insome embodiments, X₃ is A or V. In some embodiments, X₄ is A, I, or L.In some embodiments, X₅ is I, T, S, or F. In some embodiments, X₆ is E,V, or L. In some embodiments, X₇ is K or R. In some embodiments, X₈ is Eor Q. In some embodiments, X₉ is H, P, or R. In some embodiments, X₁₀ isL, T, or G. In some embodiments, X₁₁ is K or R. In some embodiments, X₁₂is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y. Insome embodiments, X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S,T, V, W, or Y. In some embodiments, X₁₄ is V or I. In some embodiments,X₁₅ is F, L, V. In some embodiments, X₁₆ is F or V.

In some embodiments, a polypeptide provided herein includes no more thanten amino acid substitutions relative to the wild-type SIRPα D1 domainhaving the sequence of SEQ ID NO: 1. In some embodiments, thepolypeptide provided herein includes no more than seven amino acidsubstitutions relative to the wild-type SIRPα D1 domain having thesequence of SEQ ID NO: 1.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of SEQ ID NO: 1. In some embodiments, the polypeptide bindsCD47 with at least 100-fold greater binding affinity than the wild-typeSIRPα D1 domain having the sequence of SEQ ID NO: 1. In someembodiments, the polypeptide binds CD47 with at least 1000-fold greaterbinding affinity than the wild-type SIRPα D1 domain having the sequenceof SEQ ID NO: 1. In some embodiments, a SIRPα D1 domain variantpolypeptide or fragment thereof binds to CD47 with a K_(D) less than1×10⁻⁸M, less than 5×10⁻⁹M, less than 1×10⁻⁹M, less 5×10⁻¹⁰M, less than1×10⁻¹⁰M or less than 1×10⁻¹¹M. In some embodiments, a SIRPα D1 domainvariant polypeptide or fragment thereof binds to CD47 with a K_(D)between about 500 nM and 100 nM, between about 100 nM and 50 nM, betweenabout 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nMand 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM.

In some embodiments, a polypeptide includes a SIRPα D1 domain varianthaving a sequence of:EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDTEX₁₆KSGAGTELSVRAKPS (SEQ ID NO: 38), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃is A or V; X₄ is V, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is S, T, or G; X₁₁ is Kor R; X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y;X₁₄ is V or I; X₁₅ is F, L, or V; and X₁₆ is F or V; and wherein thevariant comprises at least one amino acid substitution relative to awild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2.

In some embodiments in this aspect of the disclosure, a polypeptideincludes a SIRPα D1 domain variant having a sequence of SEQ ID NO: 38,wherein X₁ is L, I, or V. In some embodiments, X₂ is V, L, or, I. Insome embodiments, X₃ is A or V. In some embodiments, X₄ is V, I, or L.In some embodiments, X₅ is I, T, S, or F. In some embodiments, X₆ is E,V, or L. In some embodiments, X₇ is K or R. In some embodiments, X₈ is Eor Q. In some embodiments, X₉ is H, P, or R. In some embodiments, X₁₀ isS, T, or G. In some embodiments, X₁₁ is K or R. In some embodiments, X₁₂is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y. Insome embodiments, X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S,T, V, W, or Y. In some embodiments, X₁₄ is V or I. In some embodiments,X₁₅ is F, L, or V. In some embodiments, X₁₆ is F or V.

In some embodiments, a polypeptide includes a SIRPα D1 domain varianthaving no more than ten amino acid substitutions relative to thewild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In someembodiments, a polypeptide includes a SIRPα D1 domain variant having nomore than seven amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 2.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of SEQ ID NO: 2. In some embodiments, the polypeptide bindsCD47 with at least 100-fold greater binding affinity than the wild-typeSIRPα D1 domain having the sequence of SEQ ID NO: 2. In someembodiments, the polypeptide binds CD47 with at least 1000-fold greaterbinding affinity than the wild-type SIRPα D1 domain having the sequenceof SEQ ID NO: 2. In some embodiments, a SIRPα D1 domain variantpolypeptide or fragment thereof binds to CD47 with a K_(D) less than1×10⁻⁸M, less than 5×10⁻⁹M, less than 1×10⁻⁹M, less 5×10⁻¹⁰ in less than1×10⁻¹⁰ M or less than 1×10⁻¹¹ M. In some embodiments, a SIRPα D1 domainvariant polypeptide or fragment thereof binds to CD47 with a K_(D)between about 500 nM and 100 nM, between about 100 nM and 50 nM, betweenabout 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nMand 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM.

In another aspect, the disclosure features a polypeptide including aSIRPα D1 domain variant having a sequence of:EEX₁X₂QX₃IQPDKX₄VX₅VAAGEX₆X₇X₈LX₉CTX₁₀TSLX₁₁PVGPIQWFRGAGPX₁₂RX₁₃LIYNQX₁₄X₁₅GX₁₆FPRVTTVSX₁₇X₁₈TX₁₉RX₂₀NMDFX₂₁IX₂₂IX₂₃X₂₄ITX₂₅ADAGTYYCX₂₆KX₂₇RKGSPDX₂₈X₂₉EX₃₀KSGAGTELSVRX₃₁KPS (SEQ ID NO: 47), wherein X₁ is E or G; X₂is L, I, or V; X₃ is V, L, or, I; X₄ is S or F; X₅ is L or S; X₆ is S orT; X₇ is A or V; X₈ is I or T; X₉ is H, R, or L; X₁₀ is A, V, I, or L;X₁₁ is I, T, S, or F; X₁₂ is A or G; X₁₃ is E, V, or L; X₁₄ is K or R;X₁₅ is E or Q; X₁₆ is H, P, or R; X₁₇ is D or E; X₁₈ is S, L, T, or G;X₁₉ is K or R; X₂₀ is E or N; X₂₁ is S or P; X₂₂ is S or R; X₂₃ is S orG; X₂₄ is any amino acid; X₂₅ is any amino acid; X₂₆ is V or I; X₂₇ isF, L, V; X₂₈ is D or absent; X₂₉ is T or V; X₃₀ is F or V; and X₃₁ is Aor G; and wherein the variant comprises at least one amino acidsubstitution relative to a wild-type SIRPα D1 domain having the sequenceof SEQ ID NO: 1 or 2.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 47, wherein X₁ is E or G. In any of the aforementioned embodimentsin this aspect of the disclosure, X₂ is L, I, or V. In any of theaforementioned embodiments, X₃ is V, L, or, I. In any of theaforementioned embodiments, X₄ is S or F. In any of the aforementionedembodiments, X₅ is L or S. In any of the aforementioned embodiments, X₆is S or T. In any of the aforementioned embodiments, X₇ is A or V. Inany of the aforementioned embodiments, X₈ is I or T. In any of theaforementioned embodiments, X₉ is H or R. In any of the aforementionedembodiments, X₁₀ is A, V, I, or L. In any of the aforementionedembodiments, X₁₁ is I, T, S, or F. In any of the aforementionedembodiments, X₁₂ is A or G. In any of the aforementioned embodiments,X₁₃ is E, V, or L. In any of the aforementioned embodiments, X₁₄ is K orR. In any of the aforementioned embodiments, X₁₅ is E or Q. In any ofthe aforementioned embodiments, X₁₆ is H, P, or R. In any of theaforementioned embodiments, X₁₇ is D or E. In any of the aforementionedembodiments, X₁₈ is S, L, T, or G. In any of the aforementionedembodiments, X₁₉ is K or R. In any of the aforementioned embodiments,X₂₀ is E or N. In any of the aforementioned embodiments, X₂₁ is S or P.In any of the aforementioned embodiments, X₂₂ is S or R. In any of theaforementioned embodiments, X₂₃ is S or G. In any of the aforementionedembodiments, X₂₄ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T,V, W, or Y. In any of the aforementioned embodiments, X₂₅ is P, A, C, D,E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. In any of theaforementioned embodiments, X₂₆ is V or I. In any of the aforementionedembodiments, X₂₇ is F, L, V. In any of the aforementioned embodiments,X₂₈ is D or absent. In any of the aforementioned embodiments, X₂₉ is Tor V. In any of the aforementioned embodiments, X₃₀ is F or V. In any ofthe aforementioned embodiments, X₃₁ is A or G.

In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than ten amino acid substitutions relative to thewild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. Insome embodiments, the polypeptide of this aspect of the disclosureincludes no more than seven amino acid substitutions relative to thewild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of SEQ ID NO: 1 or 2. In some embodiments, the polypeptidebinds CD47 with at least 100-fold greater binding affinity than thewild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. Insome embodiments, the polypeptide binds CD47 with at least 1000-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of SEQ ID NO: 1 or 2. In some embodiments, a SIRPα D1 domainvariant polypeptide or fragment thereof binds to CD47 with a K_(D) lessthan 1×10⁻⁸M, less than 5×10⁻⁹M, less than 1×10⁻⁹M, less 5×10⁻¹⁰ M, lessthan 1×10⁻¹⁰ M or less than 1×10⁻¹¹M. In some embodiments, a SIRPα D1domain variant polypeptide or fragment thereof binds to CD47 with aK_(D) between about 500 nM and 100 nM, between about 100 nM and 50 nM,between about 50 nM and 10 nM, between about 10 nM and 5 nM, betweenabout 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pMand 100 pM, between about 100 pM and 50 pM, or between about 50 pM and10 pM.

In some embodiments, a polypeptide includes a SIRPα D1 domain varianthaving a sequence of:EELQX₁IQPDKSVX₂VAAGEX₃AX₄LX₅CTX₆TSLX₇PVGPIQWFRGAGPX₈RX₉LIYNQX₁₀X₁₁GX₁₂FPRVTTVSX₁₃X₁₄TKRX₁₅NMDFSIX₁₆IX₁₇X₁₈ITPADAGTYYCX₁₉KFRKGX₂₀X₂₁X₂₂DX₂₃EFKSGAGTELSVRAKPS (SEQ ID NO: 48), wherein X₁ is V or I; X₂ is L or S; X₃is T or S; X₄ is T or I; X₅ is R or H; X₆ is A, V, or I; X₇ is I, R, Y,K or F; X₈ is G or A; X₉ is E or V; X₁₀ is K or R; X₁₁ is E, D or Q; X₁₂is H or P; X₁₃ is D or E; X₁₄ is S, L or T; X₁₅ is N or E; X₁₆ is R orS; X₁₇ is G or S; X₁₈ is N or A; X₁₉ is V or I; X₂₀ is 5, I or M; X₂₁ isP or absent; X₂₂ is D or P; and X₂₃ is V or T, or a fragment thereof.

In another aspect, the disclosure features a polypeptide including aSIRPα D1 domain variant having a sequence of:EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDX₈TKRNNMDFSIRIGX₉ITPADAGTYYCX₁₀KFRKGSPDDVEFKSGAGTELSVRAK PS (SEQID NO: 49), wherein X₁ is V, L, or I; X₂ is A, I, V, or L; X₃ is I, F,S, or T; X₄ is E, V, or L; X₅ is K or R; X₆ is E or Q; X₇ is H, P, or R;X₈ is L, T, S, or G; X₉ is A; and X₁₀ is V or I; and wherein the variantcomprises at least one amino acid substitution relative to a wild-typeSIRPα D1 domain having the sequence of SEQ ID NO: 1.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 49, wherein X₁ is V, L or I. In any of the aforementionedembodiments in this aspect of the disclosure, X₂ is A, I, V, or L. Inany of the aforementioned embodiments, X₃ is I, F, S, or T. In any ofthe aforementioned embodiments, X₄ is E, V, or L. In any of theaforementioned embodiments, X₅ is K or R. In any of the aforementionedembodiments, X₆ is E or Q. In any of the aforementioned embodiments, X₇is H, P, or R. In any of the aforementioned embodiments, X₈ is L, T, Sor G. In any of the aforementioned embodiments, X₉ is A. In any of theaforementioned embodiments, X₁₀ is V or I.

In some embodiments, the polypeptide comprises a SIRPα D1 domain thatcomprises at least 85% sequence identity (e.g., at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity) to SEQ ID NO: 49, wherein each of X₁, X₂, X₃, X₄, X₅, X₆, X₇,X₈, X₉, and X₁₀ are not a wild-type amino acid.

In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than ten amino acid substitutions relative to thewild-type SIRPα D1 domain having the sequence of any one of SEQ IDNO: 1. In some embodiments, the polypeptide of this aspect of thedisclosure includes no more than seven amino acid substitutions relativeto the wild-type SIRPα D1 domain having the sequence of any one of SEQID NO: 1.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of SEQ ID NO: 1. In some embodiments, the polypeptide bindsCD47 with at least 100-fold greater binding affinity than the wild-typeSIRPα D1 domain having the sequence of SEQ ID NO: 1. In someembodiments, the polypeptide binds CD47 with at least 1000-fold greaterbinding affinity than the wild-type SIRPα D1 domain having the sequenceof SEQ ID NO: 1. In some embodiments, a SIRPα D1 domain variantpolypeptide or fragment thereof binds to CD47 with a K_(D) less than1×10⁻⁸M, less than 5×10⁻⁹M, less than 1×10⁻⁹M, less 5×10⁻¹⁰ M, less than1×10⁻¹⁰ M or less than 1×10⁻¹¹M. In some embodiments, a SIRPα D1 domainvariant polypeptide or fragment thereof binds to CD47 with a K_(D)between about 500 nM and 100 nM, between about 100 nM and 50 nM, betweenabout 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nMand 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM.

In another aspect, the disclosure features a polypeptide including aSIRPα D1 domain variant having a sequence of:EEELQX₁IQPDKSVSVAAGESAILHCTX₂TSLX₃PVGPIQWFRGAGPARX₄LIYNQX₅X₆GX₇FPRVTTVSEX₈TKRENMDFSISISX₉ITPADAGTYYCX₁₀KFRKGSPDTEFKSGAGTELSVRAKPS, (SEQID NO: 50), wherein X₁ is V or I; X₂ is V or I; X₃ is I or F; X₄ is E orV; X₅ is K or R; X₆ is E or Q; X₇ is H or P; X₈ is S or T; X₉ is N or A;and X₁₀ V or I; and wherein the variant comprises at least one aminoacid substitution relative to a wild-type SIRPα D1 domain having thesequence of SEQ ID NO: 2.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 50, wherein X₁ is V or I. In any of the aforementioned embodimentsin this aspect of the disclosure, X₂ is V or I. In any of theaforementioned embodiments, X₃ is I or F. In any of the aforementionedembodiments, X₄ is E or V. In any of the aforementioned embodiments, X₅is K or R. In any of the aforementioned embodiments, X₆ is E or Q. Inany of the aforementioned embodiments, X₇ is H or P. In any of theaforementioned embodiments, X₈ is S or R. In any of the aforementionedembodiments, X₉ is N or A. In any of the aforementioned embodiments, X₁₀is V or I.

In some embodiments, the polypeptide comprises a SIRPα D1 domain thatcomprises at least 85% sequence identity (e.g., at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity) to SEQ ID NO: 50, wherein each of X₁, X₂, X₃, X₄, X₅, X₆, X₇,X₈, X₉, and X₁₀ is not a wild-type amino acid.

In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than ten amino acid substitutions relative to thewild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In someembodiments, the polypeptide of this aspect of the disclosure includesno more than seven amino acid substitutions relative to the wild-typeSIRPα D1 domain having the sequence of SEQ ID NO: 2.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of SEQ ID NO: 2. In some embodiments, the polypeptide bindsCD47 with at least 100-fold greater binding affinity than the wild-typeSIRPα D1 domain having the sequence of SEQ ID NO: 2. In someembodiments, the polypeptide binds CD47 with at least 1000-fold greaterbinding affinity than the wild-type SIRPα D1 domain having the sequenceof SEQ ID NO: 2. In some embodiments, a SIRPα D1 domain variantpolypeptide or fragment thereof binds to CD47 with a K_(D) less than1×10⁻⁸M, less than 5×10⁻⁹M, less than 1×10⁻⁹M, less 5×10⁻¹⁰ M, less than1×10⁻¹⁰ M or less than 1×10⁻¹¹M. In some embodiments, a SIRPα D1 domainvariant polypeptide or fragment thereof binds to CD47 with a K_(D)between about 500 nM and 100 nM, between about 100 nM and 50 nM, betweenabout 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nMand 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM.

In another aspect, the disclosure features a polypeptide including aSIRPα D1 domain variant having a sequence of:EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅EGX₆FPRVTTVSDX₇TKRNNMDFSIRIGX₈ITPADAGTYYCX₉KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ IDNO: 51), wherein X₁ is V or I; X₂ is A or I; X₃ is I or F; X₄ is E or V;X₅ is K or R; X₆ is H or P; X₇ is L or T; X₈ is N or A; and X₉ is V orI; and wherein the variant comprises at least one amino acidsubstitution relative to a wild-type SIRPα D1 domain having the sequenceof SEQ ID NO: 1.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 51, wherein X₁ is V or I. In any of the aforementioned embodimentsin this aspect of the disclosure, X₂ is A or I. In any of theaforementioned embodiments, X₃ is I or F. In any of the aforementionedembodiments, X₄ is E or V. In any of the aforementioned embodiments, X₅is K or R. In any of the aforementioned embodiments, X₆ is H or P. Inany of the aforementioned embodiments, X₇ is L or T. In any of theaforementioned embodiments, X₈ is N or A. In any of the aforementionedembodiments, X₉ is V or I. In some embodiments, X₄ is not V.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 51, wherein X₈ is A. In any of the aforementioned embodiments inthis aspect of the disclosure, X₈ is A and X₁ is V or I. In any of theaforementioned embodiments in this aspect of the disclosure, X₈ is A andX₂ is A or I. In any of the aforementioned embodiments, X₈ is A and X₃is I or F. In any of the aforementioned embodiments, X₈ is A and X₄ is Eor V. In some embodiments, X₄ is not V. In any of the aforementionedembodiments, X₈ is A and X₅ is K or R. In any of the aforementionedembodiments, X₈ is A and X₆ is H or P. In any of the aforementionedembodiments, X₈ is A and X₇ is A or V. In any of the aforementionedembodiments, X₈ is A and X₉ is V or I.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 51, wherein X₈ is A. In any of the aforementioned embodiments inthis aspect of the disclosure, X₈ is A and X₁ is I. In any of theaforementioned embodiments in this aspect of the disclosure, X₈ is A andX₂ is I. In any of the aforementioned embodiments, X₈ is A and X₃ is F.In any of the aforementioned embodiments, X₈ is A and X₄ is V. In any ofthe aforementioned embodiments, X₈ is A and X₅ is R. In any of theaforementioned embodiments, X₈ is A and X₆ is P. In any of theaforementioned embodiments, X₈ is A and X₇ is T. In any of theaforementioned embodiments, X₈ is A and X₉ is I.

In some embodiments, the polypeptide comprises a SIRPα D1 domain variantthat comprises at least 85% sequence identity (e.g., at least 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity) to SEQ ID NO: 51, wherein each of X₁, X₂, X₃, X₄, X₅,X₆, X₇, X₈, and X₉ is not a wild-type amino acid.

In some embodiments, the polypeptide of this aspect of the disclosurecomprises no more than ten amino acid substitutions relative to thewild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In someembodiments, the polypeptide of this aspect of the disclosure comprisesno more than seven amino acid substitutions relative to the wild-typeSIRPα D1 domain having the sequence of SEQ ID NO: 1.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of SEQ ID NO: 1. In some embodiments, the polypeptide bindsCD47 with at least 100-fold greater binding affinity than the wild-typeSIRPα D1 domain having the sequence of SEQ ID NOs: 1. In someembodiments, the polypeptide binds CD47 with at least 1000-fold greaterbinding affinity than the wild-type SIRPα D1 domain having the sequenceof SEQ ID NO: 1. In some embodiments, a SIRPα D1 domain variantpolypeptide or fragment thereof binds to CD47 with a K_(D) less than1×10⁻⁸M, less than 5×10⁻⁹M, less than 1×10⁻¹⁰ M, less 5×10⁻¹⁰ M, lessthan 1×10⁻¹⁰ M or less than 1×10⁻¹¹ M. In some embodiments, a SIRPα D1domain variant polypeptide or fragment thereof binds to CD47 with aK_(D) between about 500 nM and 100 nM, between about 100 nM and 50 nM,between about 50 nM and 10 nM, between about 10 nM and 5 nM, betweenabout 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pMand 100 pM, between about 100 pM and 50 pM, or between about 50 pM and10 pM.

In another aspect, the disclosure features a polypeptide including aSIRPα D1 domain variant having a sequence of:EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRELIYNQX₄EGX₅FPRVTTVSDX₆TKRNNMDFSIRIGX₇ITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ IDNO: 222), wherein X₁ is V, L, or I; X₂ is A, I, or L; X₃ is I, T, S, orF; X₄ is K or R; X₅ is H or P; X₆ is L, T, or G; X₇ is N or A; andwherein the variant comprises at least one amino acid substitutionrelative to a wild-type SIRPα D1 domain having a sequence according toSEQ ID NO: 1.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 222, wherein X₁ is V, L, or I. In any of the aforementionedembodiments in this aspect of the disclosure, X₂ is A, I, or L. In anyof the aforementioned embodiments, X₃ is I, T, S, or F. In any of theaforementioned embodiments, X₄ is K or R. In any of the aforementionedembodiments, X₅ is H or P. In any of the aforementioned embodiments, X₆is L, T, or G. In any of the aforementioned embodiments, X₇ is N or A.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 222, wherein X₁ is V or I. In any of the aforementioned embodimentsin this aspect of the disclosure, X₂ is A or I. In any of theaforementioned embodiments, X₃ is I or F. In any of the aforementionedembodiments, X₄ is K or R. In any of the aforementioned embodiments, X₅is H or P. In any of the aforementioned embodiments, X₆ is L or T. Inany of the aforementioned embodiments, X₇ is N or A.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 222, wherein X₇ is A. In any of the aforementioned embodiments inthis aspect of the disclosure, X₇ is A and X₁ is V or I. In any of theaforementioned embodiments in this aspect of the disclosure, X₇ is A andX₂ is A or I. In any of the aforementioned embodiments, X₇ is A and X₃is I or F. In any of the aforementioned embodiments, X₇ is A and X₄ is Kor R. In any of the aforementioned embodiments, X₇ is A and X₅ is H orP. In any of the aforementioned embodiments, X₇ is A and X₆ is L or T.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 222, wherein X₇ is A. In any of the aforementioned embodiments inthis aspect of the disclosure, X₇ is A and X₁ is I. In any of theaforementioned embodiments in this aspect of the disclosure, X₇ is A andX₂ is I. In any of the aforementioned embodiments, X₇ is A and X₃ is F.In any of the aforementioned embodiments, X₇ is A and X₄ is R. In any ofthe aforementioned embodiments, X₇ is A and X₅ is P. In any of theaforementioned embodiments, X₇ is A and X₆ is T.

In some embodiments, the polypeptide comprises a SIRPα D1 domain thatcomprises at least 85% sequence identity (e.g., at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity) to SEQ ID NO: 222, wherein each of X₁, X₂, X₃, X₄, X₅, X₆, andX₇ is not a wild-type amino acid.

In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than ten amino acid substitutions relative to thewild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In someembodiments, the polypeptide of this aspect of the disclosure includesno more than seven amino acid substitutions relative to the wild-typeSIRPα D1 domain having the sequence of SEQ ID NO: 1.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of SEQ ID NO: 1. In some embodiments, the polypeptide bindsCD47 with at least 100-fold greater binding affinity than the wild-typeSIRPα D1 domain having the sequence of SEQ ID NO: 1. In someembodiments, the polypeptide binds CD47 with at least 1000-fold greaterbinding affinity than the wild-type SIRPα D1 domain having the sequenceof SEQ ID NO: 1. In some embodiments, fragments include polypeptides ofless than 10 amino acids in length, about 10 amino acids in length,about 20 amino acids in length, about 30 amino acids in length, about 40amino acids in length, about 50 amino acids in length, about 60 aminoacids in length, about 70 amino acids in length, about 80 amino acids inlength, about 90 amino acids in length, about 100 amino acids in length,or more than about 100 amino acids in length. Fragments retain theability to bind to CD47. Preferably, SIRPα D1 domain variantpolypeptides and fragments thereof bind to CD47 with a higher affinitythan a SIRPα polypeptide binds to CD47. For example, in someembodiments, a SIRPα D1 domain variant polypeptide or fragment thereofbinds to CD47 with a K_(D) less than 1×10⁻⁸M, less than 5×10⁻⁹M, lessthan 1×10⁻⁹ M, less 5×10⁻¹⁰ M, less than 1×10⁻¹⁰ M or less than1×10⁻¹¹M. In some embodiments, a SIRPα D1 domain variant polypeptide orfragment thereof binds to CD47 with a K_(D) between about 500 nM and 100nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM,between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pMand 50 pM, or between about 50 pM and 10 pM.

In another aspect, the disclosure features a polypeptide including aSIRPα D1 domain variant having a sequence of:EEELQX₁IQPDKSVSVAAGESAILHCTX₂TSLX₃PVGPIQWFRGAGPARELIYNQX₄EGX₅FPRVTTVSEX₆TKRENMDFSISISX₇ITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS (SEQ ID NO:212), wherein X₁ is V, L, or I; X₂ is V, I, or L; X₃ is I, T, S, or F;X₄ is K or R; X₅ is H, P, or R; X₆ is S, T, or G; X₇ is N or A; andwherein the variant comprises at least one amino acid substitutionrelative to a wild-type SIRPα D1 domain having the sequence of SEQ IDNO: 2.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 212, wherein X₁ is V, L, or I. In any of the aforementionedembodiments in this aspect of the disclosure, X₂ is V, I, or L. In anyof the aforementioned embodiments, X₃ is I, T, S, or F. In any of theaforementioned embodiments, X₄ is K or R. In any of the aforementionedembodiments, X₅ is H or P. In any of the aforementioned embodiments, X₆is S, T, or G. In any of the aforementioned embodiments, X₇ is N or A.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 212, wherein X₁ is V or I. In any of the aforementioned embodimentsin this aspect of the disclosure, X₂ is V or I. In any of theaforementioned embodiments, X₃ is I or F. In any of the aforementionedembodiments, X₄ is K or R. In any of the aforementioned embodiments, X₅is H or P. In any of the aforementioned embodiments, X₆ is S or T. Inany of the aforementioned embodiments, X₇ is N or A.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 212, wherein X₇ is A. In any of the aforementioned embodiments inthis aspect of the disclosure, X₇ is A and X₁ is V or I. In any of theaforementioned embodiments in this aspect of the disclosure, X₇ is A andX₂ is V or I. In any of the aforementioned embodiments, X₇ is A and X₃is I or F. In any of the aforementioned embodiments, X₇ is A and X₄ is Kor R. In any of the aforementioned embodiments, X₇ is A and X₅ is H orP. In any of the aforementioned embodiments, X₇ is A and X₆ is S or T.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 212, wherein X₇ is A. In any of the aforementioned embodiments inthis aspect of the disclosure, X₇ is A and X₁ is I. In any of theaforementioned embodiments in this aspect of the disclosure, X₇ is A andX₂ is I. In any of the aforementioned embodiments, X₇ is A and X₃ is F.In any of the aforementioned embodiments, X₇ is A and X₄ is R. In any ofthe aforementioned embodiments, X₇ is A and X₅ is P. In any of theaforementioned embodiments, X₇ is A and X₆ is T.

In some embodiments, the polypeptide comprises a SIRPα D1 domain havingat least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity)to SEQ ID NO: 212, wherein each of X₁, X₂, X₃, X₄, X₅, X₆, and X₇ is nota wild-type amino acid.

In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than ten amino acid substitutions relative to thewild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In someembodiments, the polypeptide of this aspect of the disclosure includesno more than seven amino acid substitutions relative to the wild-typeSIRPα D1 domain having the sequence of SEQ ID NO: 2.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of SEQ ID NO: 2. In some embodiments, the polypeptide bindsCD47 with at least 100-fold greater binding affinity than the wild-typeSIRPα D1 domain having the sequence of SEQ ID NO: 2. In someembodiments, the polypeptide binds CD47 with at least 1000-fold greaterbinding affinity than the wild-type SIRPα D1 domain having the sequenceof SEQ ID NO: 2. In some embodiments, fragments include polypeptides ofless than 10 amino acids in length, about 10 amino acids in length,about 20 amino acids in length, about 30 amino acids in length, about 40amino acids in length, about 50 amino acids in length, about 60 aminoacids in length, about 70 amino acids in length, about 80 amino acids inlength, about 90 amino acids in length, about 100 amino acids in length,or more than about 100 amino acids in length. Fragments retain theability to bind to CD47. Preferably, SIRPα D1 domain variantpolypeptides and fragments thereof bind to CD47 with a higher affinitythan a SIRPα polypeptide binds to CD47. For example, in someembodiments, a SIRPα D1 domain variant polypeptide or fragment thereofbinds to CD47 with a K_(D) less than 1×10⁻⁸M, less than 5×10⁻⁹M, lessthan 1×10⁻⁹M, less 5×10⁻¹⁰ M, less than 1×10⁻¹⁰ M or less than 1×10⁻¹¹M. In some embodiments, a SIRPα D1 domain variant polypeptide orfragment thereof binds to CD47 with a K_(D) between about 500 nM and 100nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM,between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pMand 50 pM, or between about 50 pM and 10 pM.

Described herein, in some embodiments, is a polypeptide comprising aSIRPα D1 domain variant having a sequence according to:EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDX₈TKRNNMDFSIRIGX₉X₁₀X₁₁X₁₂ADAGTYYCX₁₃KFRKGSPDDVEFKSGAGTELS VRAKPS(SEQ ID NO: 218), wherein X₁ is V, L, or I; X₂ is A, V, L, or I; X₃ isI, S, T, or F; X₄ is E, L, or V; X₅ is K or R; X₆ is E or Q; X₇ is H, R,or P; X₈ is S, G, L, or T; X₉ is any amino acid; X₁₀ is any amino acid;X₁₁ is any amino acid; X₁₂ is any amino acid; and X₁₃ is V or I; andwherein the SIRPα D1 domain variant comprises at least two amino acidsubstitutions relative to a wild-type SIRPα D1 domain having a sequenceaccording to SEQ ID NO: 1.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 212, wherein X₁, wherein X₉ is A. In any of the aforementionedembodiments in this aspect of the disclosure, X₉ is N. In any of theaforementioned embodiments in this aspect of the disclosure X₁₀ is I. Inany of the aforementioned embodiments in this aspect of the disclosureX₉ is N and X₁₀ is P. In any of the aforementioned embodiments in thisaspect of the disclosure X₉ is N and X₁₁ is any amino acid other than S,T, or C. In any of the aforementioned embodiments in this aspect of thedisclosure X₁₁ is T. In any of the aforementioned embodiments in thisaspect of the disclosure X₁₁ is an amino acid other than T. In any ofthe aforementioned embodiments in this aspect of the disclosure X₁₂ isP. In any of the aforementioned embodiments in this aspect of thedisclosure X₉ is N and X₁₂ is any amino acid other than P.

Described herein, in some embodiments, is a polypeptide comprising aSIRPα D1 domain variant having a sequence according to:EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDXsTKRNNMDFSIRIGX₉ITX₁₀ADAGTYYCX₁₁KFRKGSPDDVEFKSGAGTELSVRA KPS(SEQ ID NO: 219), wherein X₁ is V, L, or I; X₂ is A, V, L, or I; X₃ isI, S, T, or F; X₄ is E, L, or V; X₅ is K or R; X₆ is E or Q; X₇ is H, R,or P; X₈ is S, G, L, or T; X₉ is N; X₁₀ is any amino acid other than P;and X₁₁ is V or I; and wherein the SIRPα D1 domain variant comprises atleast two amino acid substitutions relative to a wild-type SIRPα D1domain having a sequence according to SEQ ID NO: 1.

In another aspect of the disclosure, compositions are disclosed hereinwhich include a SIRPα D1 domain variant polypeptide having the aminoacid sequence of SEQ ID NO: 48, or a fragment thereof. In someembodiments, the SIRPα D1 domain variant polypeptide or fragment thereofbinds to CD47 with a higher affinity compared to the affinity that aSIRPα polypeptide binds to the CD47. In some embodiments, the SIRPα D1domain variant polypeptide binds to CD47 with a K_(D) less than 1×10⁻⁸M,or less than 1×10⁻⁹M, less than 1×10⁻¹⁰ M or less than 1×10⁻¹¹ M. Insome embodiments, the above-mentioned SIRPα D1 domain variantpolypeptides are attached or fused to a second polypeptide. In someembodiments, the second polypeptide includes, without limitation, an Fcpolypeptide, an Fc variant or a fragment of the foregoing.

Without limiting the foregoing, in some embodiments, a SIRPα D1 domainvariant polypeptide is selected from any one of SEQ ID NOs: 53-87 and213 shown in Table 6.

TABLE 6 SIRPa Variant Polypeptides SEQ ID NO: Amino Acid Sequence 53EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAG TELSVRAKPS 54EEELQVIQPDKSVSVAAGESAILHCTVTSLFPVGPIQWFRGAGPARELIYNQRQGPFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSG AGTELSVRAKPS 55EEELQVIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGA GTELSVRAKPS 56EEELQIIQPDKSVSVAAGESAILHCTVTSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGA GTELSVRAKPS 57EEELQIIQPDKSVSVAAGESAILHCTITSLIPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAG TELSVRAKPS 58EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARELIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAG TELSVRAKPS 59EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQKQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAG TELSVRAKPS 60EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQREGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAG TELSVRAKPS 61EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGHFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGA GTELSVRAKPS 62EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSESTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAG TELSVRAKPS 63EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGA GTELSVRAKPS 64EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQREGPFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSG AGTELSVRAKPS 65EEELQVIQPDKSVSVAAGESAILHCTVTSLFPVGPIQWFRGAGPARELIYNQREGPFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSG AGTELSVRAKPS 66EEELQVIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARELIYNQREGPFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSG AGTELSVRAKPS 67EEELQVIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARELIYNQREGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSG AGTELSVRAKPS 68EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARELIYNQREGPFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGA GTELSVRAKPS 69EEELQVIQPDKSVSVAAGESAILHCTITSLIPVGPIQWFRGAGPARELIYNQREGPFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGA GTELSVRAKPS 70EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARELIYNQREGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGA GTELSVRAKPS 71EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEF KSGAGTELSVRAKPS 72EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKS GAGTELSVRAKPS 73EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFK SGAGTELSVRAKPS 74EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKS GAGTELSVRAKPS 75EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFK SGAGTELSVRAKPS 76EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEF KSGAGTELSVRAKPS 77EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKS GAGTELSVRAKPS 78EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKS GAGTELSVRAKPS 79EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEF KSGAGTELSVRAKPS 80EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKS GAGTELSVRAKPS 81EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEF KSGAGTELSVRAKPS 82EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFK SGAGTELSVRAKPS 83EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKS GAGTELSVRAKPS 84EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFK SGAGTELSVRAKPS 85EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKS GAGTELSVRAKPS 86EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKS GAGTELSVRAKPS 87EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGEIFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFK SGAGTELSVRAKPS 195EEELQIIQPDKSVLVAAGETATLRCTMTSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFK SGAGTELSVRAKPS 196EEELQIIQPDKSVLVAAGETATLRCTITSLKPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFK SGAGTELSVRAKPS 197EEELQIIQPDKSVLVAAGETATLRCTITSLRPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFK SGAGTELSVRAKPS 198EEELQIIQPDKSVLVAAGETATLRCTITSLYPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFK SGAGTELSVRAKPS 199EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQRDGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFK SGAGTELSVRAKPS 200EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGIPDDVEFKSG AGTELSVRAKPS 201EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGMPDDVEFKS GAGTELSVRAKPS 202EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDVEFKSG AGTELSVRAKPS 203EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSSEPDVEFKS GAGTELSVRAKPS 204EEELQIIQPDKSVLVAAGETATLRCTITSLRPVGPIQWFRGAGPGRELIYNQRDGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFK SGAGTELSVRAKPS 205EEELQIIQPDKSVLVAAGETATLRCTITSLRPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGIPDDVEFKS GAGTELSVRAKPS 206EEELQIIQPDKSVLVAAGETATLRCTITSLRPVGPIQWFRGAGPGRELIYNQRDGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGIPDDVEFKS GAGTELSVRAKPS 207EEELQIIQPDKSVLVAAGETATLRCTITSLYPVGPIQWFRGAGPGRELIYNQRDGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFK SGAGTELSVRAKPS 208EEELQIIQPDKSVLVAAGETATLRCTITSLYPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGIPDDVEFKS GAGTELSVRAKPS 209EEELQIIQPDKSVLVAAGETATLRCTITSLYPVGPIQWFRGAGPGRELIYNQRDGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGIPDDVEFKS GAGTELSVRAKPS 210EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQRDGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGIPDDVEFKS GAGTELSVRAKPS 213EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITVADAGTYYCVKFRKGSPDDVEF KSGAGTELSVRAKPS

In some embodiments, the polypeptide comprises a SIRPα D1 domain variantthat has at least 85% sequence identity (e.g., at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity) to any variant provided in Table 6.

In some embodiments, the polypeptide comprises a SIRPα D1 domain thathas at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity) to SEQ ID NOs: 80, 81, or 85 in Table 6.

Fc Domain Variants and Fusion Polypeptides Comprising Same

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein α (SIRP-α) D1 variant comprising a SIRPα D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRPα D1 domain (e.g., a wild-type SIRPα D1domain set forth in SEQ ID NO: 1 or 2); and at least one additionalamino acid mutation relative to a wild-type SIRPα D1 domain (e.g., awild-type SIRPα D1 domain set forth in SEQ ID NO: 1 or 2) at a residueselected from the group consisting of: residue 6, residue 27, residue31, residue 47, residue 53, residue 54, residue 56, residue 66, andresidue 92.

Also disclosed herein, in some embodiments, are Fc domain variantdimers, wherein the Fc domain variant dimer comprises two Fc domainvariants, wherein each Fc domain variant independently is selected from(i) a human IgG1 Fc region consisting of mutations L234A, L235A, G237A,and N297A; (ii) a human IgG2 Fc region consisting of mutations A330S,P331S and N297A; or (iii) a human IgG4 Fc region comprising mutationsS228P, E233P, F234V, L235A, delG236, and N297A.

Antibodies that target cell surface antigens can triggerimmunostimulatory and effector functions that are associated with Fcreceptor (FcR) engagement on immune cells. There are a number of Fcreceptors that are specific for particular classes of antibodies,including IgG (gamma receptors), IgE (eta receptors), IgA (alphareceptors) and IgM (mu receptors). Binding of the Fc region to Fcreceptors on cell surfaces can trigger a number of biological responsesincluding phagocytosis of antibody-coated particles (antibody-dependentcell-mediated phagocytosis, or ADCP), clearance of immune complexes,lysis of antibody-coated cells by killer cells (antibody-dependentcell-mediated cytotoxicity, or ADCC) and, release of inflammatorymediators, placental transfer, and control of immunoglobulin production.Additionally, binding of the C1 component of complement to antibodiescan activate the complement system. Activation of complement can beimportant for the lysis of cellular pathogens. However, the activationof complement can also stimulate the inflammatory response and can alsobe involved in autoimmune hypersensitivity or other immunologicaldisorders. Variant Fc regions with reduced or ablated ability to bindcertain Fc receptors are useful for developing therapeutic antibodiesand Fc-fusion polypeptide constructs which act by targeting, activating,or neutralizing ligand functions while not damaging or destroying localcells or tissues.

In some embodiments, a SIRPα D1 polypeptide construct comprises anon-naturally occurring SIRPα D1 domain variant linked to an Fc domainvariant which forms an Fc domain having ablated or reduced effectorfunction.

In some embodiments, a Fc domain variant refers to a polypeptide chainthat includes second and third antibody constant domains (e.g., CH2 andCH3). In some embodiments, an Fc domain variant also includes a hingedomain. In some embodiments, the Fc domain variant is of anyimmunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, and IgD.Additionally, in some embodiments, an Fc domain variant is of any IgGsubtype (e.g., IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3, and IgG4). In someembodiments, an Fc domain variant comprises as many as ten amino acidmodifications (e.g., insertions, deletions and/or substitutions)relative to a wild-type Fc domain monomer sequence (e.g., 1-10, 1-8,1-6, 1-4 amino acid substitutions, additions or insertions, deletions,or combinations thereof) that alter the interaction between an Fc domainand an Fc receptor.

As used herein, the term “Fc domain dimer” refers to a dimer of two Fcdomains. In a wild-type Fc domain dimer, two wild-type Fc domainsdimerize by the interaction between the two CH3 antibody constantdomains, as well as one or more disulfide bonds that form between thehinge domains of the two dimerized Fc domains.

As used herein, the term “Fc domain dimer variant” comprises at leastone Fc domain variant. In some embodiments, an Fc domain dimer variantcomprises Fc domain variants that are mutated to lack effectorfunctions, for example a “dead Fc domain dimer variant.” In someembodiments, each of the Fc domains in an Fc domain dimer variantincludes amino acid substitutions in the CH2 antibody constant domain toreduce the interaction or binding between the Fc domain dimer variantand an Fc receptor, such as an Fcγ receptor (FcγR), an Fcα receptor(FcαR), or an Fcε (FcεR).

In some embodiments, a SIRPα D1 domain variant (e.g., any of thevariants described in Tables 2, 5, and 6) is fused to an Fc domainvariant of an immunoglobulin or a fragment of an Fc domain variant. Insome embodiments, an Fc domain variant of an immunoglobulin or afragment of an Fc domain variant is capable of forming an Fc domaindimer with another Fc domain variant. In some embodiments, an Fc domainvariant of an immunoglobulin or a fragment of an Fc domain variant isnot capable of forming an Fc domain dimer with another Fc domainvariant. In some embodiments, an Fc domain variant or a fragment of anFc domain variant is fused to a polypeptide of the disclosure toincrease serum half-life of the polypeptide. In some embodiments, an Fcdomain variant or a fragment of an Fc domain variant fused to apolypeptide of the disclosure dimerizes with a second Fc domain variantto form an Fc domain dimer variant which binds an Fc receptor, oralternatively, an Fc domain variant binds to an Fc receptor. In someembodiments, an Fc domain variant or a fragment of the Fc domain variantfused to a polypeptide to increase serum half-life of the polypeptidedoes not induce any immune system-related response.

In some embodiments, a SIRPα polypeptide or construct provided hereinincludes a SIRPα D1 domain or variant thereof joined to a first Fcdomain variant and an antibody variable domain joined to a second Fcdomain variant, in which the first and second Fc domain variants combineto form an Fc domain dimer variant (e.g., a heterodimeric Fc domaindimer variant). An Fc domain dimer is the protein structure that isfound at the C-terminus of an immunoglobulin. An Fc domain dimerincludes two Fc domains that are dimerized by the interaction betweenthe CH3 antibody constant domains. A wild-type Fc domain dimer forms theminimum structure that binds to an Fc receptor, e.g., FcγRI, FcγRIIa,FcγRIIb, FcγRIIIa, FcγRIIIb, and FcγRIV.

The Fc domain dimer is not involved directly in binding an antibody toits target, but can be involved in various effector functions, such asparticipation of the antibody in antibody-dependent cellular toxicity.In some embodiments, the Fc domain in a SIRPα polypeptide or constructof the disclosure comprises amino acid substitutions, additions orinsertions, deletions, or any combinations thereof that lead todecreased effector function such as decreased antibody-dependentcell-mediated cytotoxicity (ADCC), decreased complement-dependentcytolysis (CDC), decreased antibody-dependent cell-mediated phagocytosis(ADCP), or any combinations thereof. In some embodiments, the SIRPαpolypeptides or constructs of the disclosure are characterized bydecreased binding (e.g., minimal binding or absence of binding) to ahuman Fc receptor and decreased binding (e.g., minimal binding orabsence of binding) to complement protein C1q. In some embodiments, theSIRPα constructs of the disclosure are characterized by decreasedbinding (e.g., minimal binding or absence of binding) to human FcγRI,FcγRIIA, FcγRIIB, FcγRIIIB, or any combinations thereof, and C1q. Toalter or reduce an antibody-dependent effector function, such as ADCC,CDC, ADCP, or any combinations thereof, in some embodiments, the Fcdomains in SIRPα constructs of the disclosure are of the IgG class andcomprise one or more amino acid substitutions at E233, L234, L235, G236,G237, D265, D270, N297, E318, K320, K322, A327, A330, P331, or P329(numbering according to the EU index of Kabat (Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991))).

In some embodiments, polypeptide constructs comprising a non-native Fcregion described herein exhibit reduced or ablated binding to at leastone of Fcγ receptors CD16a, CD32a, CD32b, CD32c, and CD64 as compared toa polypeptide construct comprising a native Fc region. In some cases,the polypeptide constructs described herein exhibit reduced or ablatedbinding to CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors.

CDC refers to a form of cytotoxicity in which the complement cascade isactivated by the complement component C1q binding to antibody Fcdomains. In some embodiments, polypeptide constructs comprising anon-native Fc region described herein exhibit at least a 5%, 10%, 15%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in C1qbinding compared to a polypeptide construct comprising a wild-type Fcregion. In some cases, polypeptide constructs comprising a non-native Fcregion as described herein exhibit reduced CDC as compared to apolypeptide construct comprising a wild-type Fc region. In someembodiments, polypeptide constructs comprising a non-native Fc region asdescribed herein exhibit at least a 5%, 10%, 15%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or greater reduction in CDC compared to a polypeptideconstruct comprising a wild-type Fc region. In some cases, polypeptideconstructs comprising a non-natural Fc domain variants or Fc domaindimer variants as described herein exhibit negligible CDC as compared toa polypeptide construct comprising a wild-type Fc region.

In some embodiments, the Fc domain variants or Fc domain dimer variantsdescribed herein are minimally glycosylated or have reducedglycosylation relative to a wild-type sequence. In some embodiments,deglycosylation is accomplished with a mutation of N297A, or by mutatingN297 to any amino acid which is not N. In some embodiments,deglycosylation is accomplished by disrupting the motifN-Xaa1-Xaa2-Xaa3, wherein N=asparagine; Xaa1=any amino acid except P(proline); Xaa2=T (threonine), S (serine) or C (cysteine); and Xaa3=anyamino acid except P (proline). In one embodiment, the N-Xaa1-Xaa2-Xaa3motif refers to residues 297-300 as designated according to Kabat etal., 1991. In some embodiments, a mutation to any one or more of N,Xaa1, Xaa2, or Xaa3 results in deglycosylation of the Fc domain variantor Fc domain dimer variant.

In some embodiments, variants of antibody IgG constant regions (e.g., Fcdomain variants or Fc domain dimer variants) possess a reduced capacityto specifically bind Fcγ receptors or have a reduced capacity to inducephagocytosis. In some embodiments, variants of antibody IgG constantregions (e.g., Fc domain variants or Fc domain dimer variants) possess areduced capacity to specifically bind Fcγ receptors and have a reducedcapacity to induce phagocytosis. For example, in some embodiments, an Fcdomain variant is mutated to lack effector functions, typical of a“dead” Fc domain variant. For example, in some embodiments, an Fc domainvariant includes specific amino acid substitutions that are known tominimize the interaction between the Fc domain dimer and an Fcγreceptor. In some embodiments, an Fc domain variant is from an IgG1antibody and includes one or more of amino acid substitutions L234A,L235A, G237A, and N297A (as designated according to the EU numberingsystem per Kabat et al., 1991). In some embodiments, one or moreadditional mutations are included in such IgG1 Fc domain variant.Non-limiting examples of such additional mutations for human IgG1 Fcdomain variants include E318A and K322A. In some instances, a human IgG1Fc domain variant has up to 12, 11, 10, 9, 8, 7, 6, 5 or 4 or fewermutations in total as compared to wild-type human IgG1 sequence. In someembodiments, one or more additional deletions are included in such IgG1Fc domain variant. For example, in some embodiments, the C-terminallysine of the Fc domain IgG1 heavy chain constant region provided in SEQID NO: 88 in Table 7 is deleted, for example to increase the homogeneityof the polypeptide when the polypeptide is produced in bacterial ormammalian cells. In some instances, a human IgG1 Fc domain variant hasup to 12, 11, 10, 9, 8, 7, 6, 5 or 4 or fewer deletions in total ascompared to wild-type human IgG1 sequence (see, e.g., SEQ ID NO: 161below). In some embodiments, a IgG1 Fc domain variant has a sequenceaccording to any one of SEQ ID NO: 135, SEQ ID NO: 136 or SEQ ID NO:137.

SEQ ID NO: 161: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

In some embodiments, an Fc domain variant is from an IgG2 or IgG4antibody and includes amino acid substitutions A330S, P331S, or bothA330S and P331S. The aforementioned amino acid positions are definedaccording to Kabat, et al. (1991). The Kabat numbering of amino acidresidues can be determined for a given antibody by alignment at regionsof homology of the sequence of the antibody with a “standard” Kabatnumbered sequence. In some embodiments, the Fc domain variant comprisesa human IgG2 Fc domain sequence comprising one or more of A330S, P331Sand N297A amino acid substitutions (as designated according to the EUnumbering system per Kabat, et al. (1991). In some embodiments, one ormore additional mutations are included in such IgG2 Fc domain variants.Non-limiting examples of such additional mutations for human IgG2 Fcdomain variant include V234A, G237A, P238S, V309L and H268A (asdesignated according to the EU numbering system per Kabat et al.(1991)). In some instances, a human IgG2 Fc domain variant has up to 12,11, 10, 9, 8, 7, 6, 5, 4, 3 or fewer mutations in total as compared towild-type human IgG2 sequence. In some embodiments, one or moreadditional deletions are included in such IgG2 Fc domain variant. Forexample, in some embodiments, the C-terminal lysine of the Fc domainIgG2 heavy chain constant region provided in SEQ ID NO: 89 in Table 7 isdeleted, for example to increase the homogeneity of the polypeptide whenthe polypeptide is produced in bacterial or mammalian cells. In someinstances, a human IgG2 Fc domain variant has up to 12, 11, 10, 9, 8, 7,6, 5 or 4 or fewer deletions in total as compared to wild-type humanIgG2 sequence (see, e.g., SEQ ID NO: 162 below).

SEQ ID NO: 162: ERKCCVECPPCPAPPVAGPSVFLFPFKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNTQKSLSLSPG

When the Fc domain variant is an IgG4 Fc domain variant, in someembodiments, such Fc domain variant comprises a S228P mutation (asdesignated according to Kabat, et al. (1991)). In some instances, ahuman IgG4 Fc domain variant has up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2 or 1 mutation(s) in total as compared to wild-type human IgG4sequence. In some embodiments, the Fc domain variant comprises a humanIgG4 Fc sequence comprising one or more of S228P, E233P, F234V, L235A,and delG236 amino acid substitutions (as designated according to the EUnumbering system per Kabat, et al. (1991). In some embodiments, the Fcdomain variant comprises a human IgG4 Fc sequence comprising one or moreof S228P, E233P, F234V, L235A, delG236, and N297A amino acidsubstitutions (as designated according to the EU numbering system perKabat, et al. (1991).

In some embodiments, the Fc domain variant includes at least one of themutations L234A, L235A, G237A or N297A of an IgG1 Fc region or at leastone of the mutations A330S, P331S or N297A of an IgG2 Fc region. In someembodiments, the Fc domain variant includes at least two of themutations L234A, L235A, G237A or N297A of an IgG1 Fc region or at leasttwo of the mutations A330S, P331S or N297A of an IgG2 Fc region. In someembodiments, the Fc domain variant includes at least three of themutations L234A, L235A, G237A or N297A of an IgG1 Fc region or consistsof the mutations A330S, P331S and N297A of an IgG2 Fc region. In someembodiments, the Fc domain variant consists of the mutations L234A,L235A, G237A and N297A.

In some embodiments, the Fc domain variant exhibits reduced binding toan Fc receptor of the subject compared to the wild-type human IgG Fcregion. In some embodiments, the Fc domain variant exhibits ablatedbinding to an Fc receptor of the subject compared to the wild-type humanIgG Fc region. In some embodiments, the Fc domain variant exhibits areduction of phagocytosis compared to the wild-type human IgG Fc region.In some embodiments, the Fc domain variant exhibits ablated phagocytosiscompared to the wild-type human IgG Fc region.

SEQ ID NO: 88 and SEQ ID NO: 89 provide amino acid sequences of Fcdomain IgG1 and IgG2 heavy chain constant regions. In some embodiments,an Fc domain variant is any variant of SEQ ID NOs: 90-95 as shown inTable 7.

TABLE 7 Amino Acid Sequences of Fc Domain Variants SEQ ID NO:AMINO ACID SEQUENCE 88EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 89STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVIDEIKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMEI EALHNHYTQKSLSLSPGK90 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 91DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 92VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK93 VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG 94ERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVEINAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 95ERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVEINAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Antibody-dependent cell-mediated cytotoxicity, which is also referred toherein as ADCC, refers to a form of cytotoxicity in which secreted Igbound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g.,Natural Killer (NK) cells and neutrophils) enabling these cytotoxiceffector cells to bind specifically to an antigen-bearing target celland subsequently kill the target cell. Antibody-dependent cell-mediatedphagocytosis, which is also referred to herein as ADCP, refers to a formof cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain phagocytic cells (e.g., macrophages) enabling thesephagocytic effector cells to bind specifically to an antigen-bearingtarget cell and subsequently engulf and digest the target cell.Ligand-specific high-affinity IgG antibodies directed to the surface oftarget cells can stimulate the cytotoxic or phagocytic cells and can beused for such killing. In some embodiments, polypeptide constructscomprising an Fc domain variant or Fc domain dimer variant as describedherein exhibit reduced ADCC or ADCP as compared to a polypeptideconstruct comprising a wild-type Fc region. In some embodiments,polypeptide constructs comprising an Fc domain variant or Fc domaindimer variant as described herein exhibit at least a 5%, 10%, 15%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in ADCC or ADCPcompared to a polypeptide construct comprising a wild-type Fc region. Insome embodiments, polypeptide constructs comprising an Fc domain variantor Fc domain dimer variant as described herein exhibit ablated ADCC orADCP as compared to a polypeptide construct comprising a wild-type Fcregion.

Complement-directed cytotoxicity, which is also referred to herein asCDC, refers to a form of cytotoxicity in which the complement cascade isactivated by the complement component C1q binding to antibody Fcdomains. In some embodiments, polypeptide constructs comprising an Fcdomain variant or Fc domain dimer variant as described herein exhibit atleast a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greaterreduction in C1q binding compared to a polypeptide construct comprisinga wild-type Fc region. In some cases, polypeptide constructs comprisingan Fc domain variant or Fc domain dimer variant as described hereinexhibit reduced CDC as compared to a polypeptide construct comprising awild-type Fc region. In some embodiments, polypeptide constructscomprising an Fc domain variant or Fc domain dimer variant as describedherein exhibit at least a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or greater reduction in CDC compared to a polypeptide constructcomprising a wild-type Fc region. In some cases, polypeptide constructscomprising an Fc domain variant or Fc domain dimer variant as describedherein exhibit negligible CDC as compared to a polypeptide constructcomprising a wild-type Fc region.

Fc domain variants or Fc domain dimer variants herein include those thatexhibit reduced binding to an Fcγ receptor compared to the wild-typehuman IgG Fc region. For example, in some embodiments, an Fc domainvariant or Fc domain dimer variant exhibits binding to an Fcγ receptorthat is less than the binding exhibited by a wild-type human IgG Fcregion to an Fcγ receptor, as described in the Examples. In someinstances, an Fc domain variant or Fc domain dimer variant has reducedbinding to an Fcγ receptor by a factor of 10%, 20% 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (fully ablated effectorfunction). In some embodiments, the reduced binding is for any one ormore Fcγ receptor, e.g., CD16a, CD32a, CD32b, CD32c, or CD64.

In some instances, the Fc domain variants or Fc domain dimer variantsdisclosed herein exhibit a reduction of phagocytosis compared to itswild-type human IgG Fc region. Such Fc domain variants or Fc domaindimer variants exhibit a reduction in phagocytosis compared to itswild-type human IgG Fc region, wherein the reduction of phagocytosisactivity is e.g., by a factor of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 96%, 97%, 98%, 99% or 100%. In some instances, an Fc domainvariant or Fc domain dimer variant exhibits ablated phagocytosiscompared to its wild-type human IgG Fc region.

In some embodiments, the Fc domain variants or Fc domain dimer variantsdisclosed herein are coupled to one or more fusion partners. In somecases the fusion partner is a therapeutic moiety. In some cases, thefusion partner is selected to enable targeting of an expressed protein,purification, screening, display, and the like. In some embodiments, thefusion partner also affects the degree of binding to Fc receptors or thedegree of phagocytosis reduction. As described herein, in someembodiments, when an Fc domain variant or Fc domain dimer variant iscoupled to a fusion partner, it forms a polypeptide construct asdescribed below.

In some embodiments, fusion partners are linked to the Fc domain variantor Fc domain dimer variant sequence via a linker sequence. In someembodiments, the linker sequence generally comprises a small number ofamino acids, such as less than ten amino acids, although longer linkersare also utilized. In some cases, the linker has a length less than 10,9, 8, 7, 6, or 5 amino acids or shorter. In some cases, the linker has alength of at least 10, 11, 12, 13, 14, 15, 20, 25, 30, or 35 amino acidsor longer. Optionally, in some embodiments, a cleavable linker isemployed.

In some embodiments, a fusion partner is a targeting or signal sequencethat directs an Fc domain variant or Fc domain dimer variant protein andany associated fusion partners to a desired cellular location or to theextracellular media. In some embodiments, certain signaling sequencestarget a protein to be either secreted into the growth media, or intothe periplasmic space, located between the inner and outer membrane ofthe cell. In some embodiments, a fusion partner is a sequence thatencodes a peptide or protein that enables purification or screening.Such fusion partners include, but are not limited to, polyhistidine tags(His-tags) (for example His6 (SEQ ID NO: 223) and His10 (SEQ ID NO:224)) or other tags for use with Immobilized Metal AffinityChromatography (IMAC) systems (e.g., Ni+2 affinity columns), GSTfusions, MBP fusions, Strep-tag, the BSP biotinylation target sequenceof the bacterial enzyme BirA, and epitope tags which are targeted byantibodies (for example c-myc tags, flag-tags, and the like).

In some embodiments, such tags are useful for purification, forscreening, or both. For example, in some embodiments, an Fc domainvariant or Fc domain dimer variant is purified using a His-tag byimmobilizing it to a Ni+2 affinity column, and then after purificationthe same His-tag is used to immobilize the antibody to a Ni+2 coatedplate to perform an ELISA or other binding assay as described elsewhereherein. In some embodiments, a fusion partner enables the use of aselection method to screen Fc domain variants or Fc domain dimervariants as described herein.

Various fusion partners that enable a variety of selection methods areavailable. For example, by fusing the members of an Fc domain variant orFc domain dimer variant library to the gene III protein, phage displaycan be employed. In some embodiments, fusion partners Fc domain variantsor Fc domain dimer variants to be labeled. Alternatively, in someembodiments, a fusion partner binds to a specific sequence on theexpression vector, enabling the fusion partner and associated Fc domainvariant or Fc domain dimer variant to be linked covalently ornoncovalently with the nucleic acid that encodes them.

In some embodiments, when a fusion partner is a therapeutic moiety, thetherapeutic moiety is, e.g., a peptide, a protein, an antibody, a siRNA,or a small molecule. Non-limiting examples of therapeutic antibodiesthat are coupled to the Fc domain variants or Fc domain dimer variantsof the present disclosure include, but are not limited to antibodiesthat recognize CD47. Non-limiting examples of therapeutic polypeptidesthat are coupled to the Fc domain variants or Fc domain dimer variantsof the present disclosure include, but are not limited to, CD47 bindingpolypeptides, including SIRPα polypeptides. In such instances, the CD47binding polypeptide is attached or fused to an Fc domain variant or Fcdomain dimer variant of the disclosure. Examples of CD47 bindingpolypeptides include, but are not limited to, anti-CD47 antibodies orfragments thereof, and ligands of CD47 such as SIRPα or a fragmentthereof. Additional examples of CD47 binding polypeptides include, butare not limited to naturally-occurring forms of SIRPα as well as mutantsthereof.

In some embodiments, disclosed herein is a polypeptide comprising an Fcdomain dimer variant, wherein the Fc domain dimer variant comprises twoFc domain variants, wherein each Fc domain variant independently isselected from (i) a human IgG1 Fc region consisting of mutations L234A,L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting ofmutations A330S, P331S and N297A; or (iii) a human IgG4 Fc regioncomprising mutations S228P, E233P, F234V, L235A, delG236, and N297A. Insome embodiments, the Fc domain variants are identical (i.e.,homodimer). In some embodiments, the Fc domain variants are different(i.e., heterodimer). In some embodiments, at least one of the Fc domainvariant in an Fc domain dimer is a human IgG1 Fc region consisting ofmutations L234A, L235A, G237A, and N297A. In some embodiments, at leastone of the Fc domain variants in an Fc domain dimer is a human IgG2 Fcregion consisting of mutations A330S, P331S and N297A. In someembodiments, the Fc domain dimer variant exhibits ablated or reducedbinding to an Fcγ receptor compared to the wild-type version of thehuman IgG Fc region. In some embodiments, the Fc domain dimer variantexhibits ablated or reduced binding to CD16a, CD32a, CD32b, CD32c, andCD64 Fcγ receptors compared to the wild-type version of the human IgG Fcregion. In some embodiments, the Fc domain dimer variant exhibitsablated or reduced binding to C1q compared to the wild-type version ofthe human IgG Fc fusion. In some embodiments, at least one of the Fcdomain variants in an Fc domain dimer variant is a human IgG4 Fc regioncomprising mutations S228P, E233P, F234V, L235A, delG236, and N297A. Insome embodiments, the Fc domain dimer variant exhibits ablated orreduced binding to an Fcγ receptor compared to the wild-type human IgG4Fc region. In some embodiments, the Fc domain dimer variant exhibitsablated or reduced binding to CD16a and CD32b Fcγ receptors compared tothe wild-type version of its human IgG4 Fc region. In some embodiments,the Fc domain dimer variant binds to an Fcγ receptor with a K_(D)greater than about 5×10⁻⁶ M.

In some embodiments, the Fc domain dimer variant further comprises aCD47 binding polypeptide. In some embodiments, the Fc domain dimervariant exhibits ablated or reduced binding to an Fcγ receptor comparedto a wild-type version of a human IgG Fc region. In some embodiments,the CD47 binding polypeptide does not cause acute anemia in rodents andnon-human primates. In some embodiments, the CD47 binding polypeptidedoes not cause acute anemia in humans.

In some embodiments, the CD47 binding polypeptide is a signal-regulatoryprotein α (SIRP-α) polypeptide or a fragment thereof. In someembodiments, the SIRPα polypeptide comprises a SIRPα D1 domain variantcomprising the amino acid sequence,EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅EGX₆FPRVTTVSDX₇TKRNNMDFSIRIGX₈ITPADAGTYYCX₉KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ IDNO: 221), wherein X₁ is V or I; X₂ is A or I; X₃ is I or F; X₄ is E orV; X₅ is K or R; X₆ is H or P; X₇ is L or T; X₈ is any amino acid otherthan N; and X₉ is V or I. In some embodiments, the SIRPα polypeptidecomprises a SIRPα D1 domain variant wherein X₁ is V or I; X₂ is A or I;X₃ is I or F; X₄ is E; X₅ is K or R; X₆ is H or P; X₇ is L or T; X₈ isnot N; and X₉ is V.

In some embodiments, disclosed herein, is a polypeptide comprising: aSIRPα D1 domain variant, wherein the SIRPα D1 domain variant is anon-naturally occurring high affinity SIRPα D1 domain, wherein the SIRPαD1 domain variant binds to human CD47 with an affinity that is at least10-fold greater than the affinity of a naturally occurring D1 domain;and an Fc domain variant, wherein the Fc domain variant is linked to asecond polypeptide comprising a second Fc domain variant to form an Fcdomain dimer variant, wherein the Fc domain dimer variant has ablated orreduced effector function. In some embodiments, the non-naturallyoccurring high affinity SIRPα D1 domain comprises an amino acid mutationat residue 80.

In some embodiments, disclosed herein, is a SIRPα D1 domain variant,wherein the SIRPα D1 domain variant binds CD47 from a first species witha K_(D) less than 250 nM; and wherein the SIRPα D1 domain variant bindsCD47 from a second species with a K_(D) less than 250 nM; and the K_(D)for CD47 from the first species and the K_(D) for CD47 from the secondspecies are within 100 fold of each other; wherein the first species andthe second species are selected from the group consisting of: human,rodent, and non-human primate. In some embodiments, the SIRPα D1 domainvariant binds CD47 from at least 3 different species. In someembodiments, the non-human primate is cynomolgus monkey.

In some embodiments, disclosed herein, is a polypeptide comprising (a) aSIRPα D1 domain that binds human CD47 with a K_(D) less than 250 nM; and(b) an Fc domain or variant thereof linked to the N-terminus or theC-terminus of the SIRPα D1 domain, wherein the polypeptide does notcause acute anemia in rodents and non-human primates. In someembodiments, the polypeptide is a non-naturally occurring variant of ahuman SIRP-α. In some embodiments, administration of the polypeptide invivo results in hemoglobin reduction by less than 50% during the firstweek after administration. In some embodiments, administration of thepolypeptide in humans results in hemoglobin reduction by less than 50%during the first week after administration. In some embodiments, thepolypeptide further comprises at least one Fc domain dimer variant,wherein the Fc domain dimer variant comprises an Fc domain variantselected from (i) a human IgG1 Fc region consisting of mutations L234A,L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting ofmutations A330S, P331S and N297A; or (iii) a human IgG4 Fc regioncomprising mutations S228P, E233P, F234V, L235A, delG236, and N297A. Insome embodiments, the Fc domain variant is a human IgG1 Fc regionconsisting of mutations L234A, L235A, G237A, and N297A. In someembodiments, the Fc domain variant is a human IgG2 Fc region consistingof mutations A330S, P331S and N297A.

The SIRPα constructs of the disclosure include a SIRPα domain or variantthereof that has its C-terminus joined to the N-terminus of an Fc domainor variant thereof by way of a linker using conventional genetic orchemical means, e.g., chemical conjugation. In some embodiments, alinker (e.g., a spacer) is inserted between the polypeptide and the Fcdomain or variant thereof. In some embodiments, a polypeptide of thedisclosure including a SIRPα D1 domain variant is fused to an Fc domainvariant that is incapable of forming a dimer. In some embodiments, apolypeptide of the disclosure is fused to an Fc domain or variantthereof that is capable of forming a dimer, e.g., a heterodimer, withanother Fc domain or variant thereof. In some embodiments, a polypeptideof the invention is fused to an Fc domain or variant thereof and thisfusion protein forms a homodimer. In some embodiments, a polypeptide ofthe disclosure is fused to a first Fc domain or variant thereof and adifferent protein or peptide (e.g., an antibody variable region) isfused to a second Fc domain or variant thereof. In some embodiments, aSIRPα D1 domain or variant thereof is joined to a first Fc domain orvariant thereof and a therapeutic protein (e.g., a cytokine, aninterleukin, an antigen, a steroid, an anti-inflammatory agent, or animmunomodulatory agent) is joined to a second Fc domain or variantthereof. In some embodiments, the first and second Fc domains orvariants thereof form a heterodimer.

Without the limiting the foregoing, in some embodiments, a SIRPα D1domain variant polypeptide (e.g., any of the variants described inTables 2, 5, and 6) is fused to an Fc polypeptide or Fc variantpolypeptide, such as an Fc domain or variant thereof. Examples ofpolypeptides comprising a SIRPα D1 domain variant polypeptide and afused Fc domain variant polypeptide include, but are not limited to, SEQID NOS: 96-137, 214, and 216 shown in Table 8.

TABLE 8 Polypeptides Comprising SIRPa D1 DomainVariants Fused to Fc Domain Variants SEQ ID NO: Amino Acid Sequence 96EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 97EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 98EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 99EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 100EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 101EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVEINAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 102EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 103EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVEINAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 104EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 105EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSEIEDPEVQFNWYVDGVEVEINAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 106EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSEIEDPEVQFNWYVDGVEVEINAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 107EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSEIEDPEVQFNWYVDGVEVEINAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 108EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSEIEDPEVQFNWYVDGVEVEINAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 109EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSEIEDPEVQFNWYVDGVEVEINAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 110EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSEIEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 111EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVEINAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMITEALHNHYTQKSLSLSPGK 112EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 113EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVEINAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMITEALHNHYTQKSLSLSPGK 114EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVEINAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 115EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVEINAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 116EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVEINAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 117EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVEINAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 118EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVEINAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 119EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMITEALHNHYTQKSLSLSPGK 120EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVEINAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 121EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMITEALHNHYTQKSLSLSPGK 122EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVEINAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 123EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 124EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 125EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 126EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVEINAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 127EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVEINAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 128EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVEINAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 129EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVEINAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 130EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPSESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVEINAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 131EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVEINAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 132EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVEINAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 133EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVEINAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 134EEELQIIQPDKSVLVAAGETATLRCTIT SLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSAAAPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVEINAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK 135EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWERGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 136EEELQIIQPDKSVLVAAGETATLRCTITSLEPVGPIQWERGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 137EEELQIIQPDKSVLVAAGETATLRCTITSLEPVGPIQWERGAGPGRVLIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKERKGSPDDVEEKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 211EEELQIIQPDKSVLVAAGETATLRCTITSLRPVGPIQWFRGAGPGRELIYNQRDGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGIPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 214EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWERGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVENAKTKPREEQFASTERVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 216EEELQIIQPDKSVLVAAGETATLRCTITSLEPVGPIQWERGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKERKGSPDDVEEKSGAGTELSVRAKPSDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMBEALHNHYTQKSLSLSPGK 217EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSEKTHTCPECPAPEAAGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In some embodiments, the polypeptide comprises a SIRPα D1 variant domainthat has at least 85% sequence identity (e.g., at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity) to any variant provided in Table 8.

In some embodiments, the polypeptide comprises a SIRPα D1 domain variantthat has at least 85% sequence identity (e.g., at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity) to SEQ ID NOs: 98-104, 107-113, 116-122, or 135-137 in Table8.

In some embodiments, the polypeptide comprises (a) a signal-regulatoryprotein a (SIRP-α) D1 variant, wherein the SIRPα D1 domain variantcomprises the amino acid sequence,EEX₁X₂QX₃IQPDKX₄VX₅VAAGEX₆X₇X₈LX₉CTX₁₀TSLX₁₁PVGPIQWFRGAGPX₁₂RX₁₃LIYNQX₁₄X₁₅GX₁₆FPRVTTVSX₁₇X₁₈TX₁₉RX₂₀NMDFX₂₁IX₂₂IX₂₃X₂₄ITX₂₅ADAGTYYCX₂₆KX₂₇RKGSPDX₂₈X₂₉EX₃₀KSGAGTELSVRX₃₁KPS (SEQ ID NO: 47), wherein X₁ is E, or G; X₂is L, I, or V; X₃ is V, L, or I; X₄ is S, or F; X₅ is L, or S; X₆ is S,or T; X₇ is A, or V; X₈ is I, or T; X₉ is H, R, or L; X₁₀ is A, V, I, orL; X₁₁ is I, T, S, or F; X₁₂ is A, or G; X₁₃ is E, V, or L; X₁₄ is K, orR; X₁₅ is E, or Q; X₁₆ is H, P, or R; X₁₇ is D, or E; X₁₈ is S, L, T, orG; X₁₉ is K, or R; X₂₀ is E, or N; X₂₁ is S, or P; X₂₂ is S, or R; X₂₃is S, or G; X₂₄ is any amino acid; X₂₅ is any amino acid; X₂₆ is V, orI; X₂₇ is F, L, or V; X₂₈ is D or absent; X₂₉ is T, or V; X₃₀ is F, orV; and X₃₁ is A, or G; and wherein the SIRPα D1 domain variant comprisesat least two amino acid substitutions relative to a wild-type SIRPα D1domain having a sequence according to any one of SEQ ID NOs: 1 to 10;and (b) an Fc domain dimer variant having two Fc domain variants,wherein each Fc domain variant independently is (i) a human IgG1 Fcregion comprising a N297A mutation; (ii) a human IgG1 Fc regioncomprising L234A, L235A, and G237A mutations; (iii) a human IgG1 Fcregion comprising L234A, L235A, G237A, and N297A mutations; (iv) a humanIgG2 Fc region comprising a N297A mutation; (v) a human IgG2 Fc regioncomprising A330S and P331S mutations; (vi) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations; (vii) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, and delG236 mutations; or(viii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A,delG236, and N297A mutations.

In some embodiments, the polypeptide comprises a SIRPα D1 domain variantwherein the SIRPα D1 domain variant comprises an amino acid sequenceaccording to SEQ ID NO: 47; an Fc domain dimer having two Fc domains,wherein one of the Fc domains is an Fc domain variant comprising a humanIgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations.

Dimerization of Fc Domains

In some embodiments, a SIRPα D1 domain variant polypeptide (e.g., any ofthe variants described in Tables 2, 5, and 6) is fused to a first Fcdomain (e.g., an Fc domain variant) either at the N-terminus or at theC-terminus. In some embodiments, the first Fc domain is a variant thatis incapable of forming an dimer. In some embodiments, the first Fcdomain forms a dimer with a second Fc domain. In some embodiments, thefirst and second Fc domains comprise amino acid substitutions thatpromote heterodimerization between the first and second domain Fcdomains.

In some embodiments, each of the two Fc domains in an Fc domain dimerincludes amino acid substitutions that promote the heterodimerization ofthe two monomers. In some embodiments, a SIRPα construct is formed, forexample, from a first subunit including a SIRPα D1 domain variantpolypeptide fused to a first Fc domain and a second subunit including asecond Fc domain (e.g., without a SIRPα D1 domain variant polypeptide orany other polypeptide). In some embodiments, a construct has a singleSIRPα D1 domain variant polypeptide linked to an Fc domain dimer (e.g.,single arm). In some embodiments, a construct has two SIRPα D1 domainvariant polypeptides linked to an Fc domain dimer (e.g., double arm). Insome embodiments, a SIRPα D1 domain variant having a K_(D) of about 500nM is particularly useful in a double arm construct. In someembodiments, a SIRPα D1 domain variant having a K_(D) of about 50 nM isparticularly useful in a double arm construct. In some embodiments, aSIRPα D1 domain variant having a K_(D) of about 5 nM is useful in adouble arm construct and a single arm construct. In some embodiments, aSIRPα D1 domain variant having a K_(D) of about 500 pM is useful in adouble arm construct and a single arm construct. In some embodiments, aSIRPα D1 domain variant having a K_(D) of about 100 pM is useful in adouble arm construct and a single arm construct. In some embodiments, aSIRPα D1 domain variant having a K_(D) of about 50 pM is useful in adouble arm construct and a single arm construct. In some embodiments, aSIRPα D1 domain variant having a K_(D) of about 10 pM is useful in adouble arm construct and a single arm construct.

In some embodiments, heterodimerization of Fc domains is promoted byintroducing different, but compatible, substitutions in the two Fcdomains, such as “knob-into-hole” residue pairs and charge residuepairs. The knob and hole interaction favors heterodimer formation,whereas the knob-knob and the hole-hole interaction hinder homodimerformation due to steric clash and deletion of favorable interactions. Ahole refers to a void that is created when an original amino acid in aprotein is replaced with a different amino acid having a smallerside-chain volume. A knob refers to a bump that is created when anoriginal amino acid in a protein is replaced with a different amino acidhaving a larger side-chain volume. For example, in some embodiments, anamino acid being replaced is in the CH3 antibody constant domain of anFc domain and involved in the dimerization of two Fc domains. In someembodiments, a hole in one CH3 antibody constant domain is created toaccommodate a knob in another CH3 antibody constant domain, such thatthe knob and hole amino acids act to promote or favor theheterodimerization of the two Fc domains. In some embodiments, a hole inone CH3 antibody constant domain is created to better accommodate anoriginal amino acid in another CH3 antibody constant domain. In someembodiments, a knob in one CH3 antibody constant domain is created toform additional interactions with original amino acids in another CH3antibody constant domain.

In some embodiments, a hole is constructed by replacing amino acidshaving larger side chains such as tyrosine or tryptophan with aminoacids having smaller side chains such as alanine, valine, or threonine,for example a Y407V mutation in the CH3 antibody constant domain.Similarly, in some embodiments, a knob is constructed by replacing aminoacids having smaller side chains with amino acids having larger sidechains, for example a T366W mutation in the CH3 antibody constantdomain. In some embodiments, one Fc domain includes the knob mutationT366W and the other Fc domain includes hole mutations T366S, L358A, andY407V. In some embodiments, a polypeptide of the disclosure including aSIRPα D1 domain variant is fused to an Fc domain including the knobmutation T366W to limit unwanted knob-knob homodimer formation. Examplesof knob-into-hole amino acid pairs are included, without limitation, inTable 9 and examples of knob-into-hole Fc domain variants and SIRPα-Fcfusions are provided in Table 10.

TABLE 9 Knob-Into-Hole Amino Acid Pairs First Y407T Y407A F405A T394ST366S T394W T394S T366W Fc Domain L358A Y407T Y407A T394S Y407V SecondT366Y T366W T394W F405W T366W T366Y T366W F405W Fc Domain F405A F405WY407A

TABLE 10 Exemplary Fc Domain Variants and SIRPa D1 DomainVariant − Fc Domain Variant Fusion Polypeptides SEQ ID NO:Amino Acid Sequence 138EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 139DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 140EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 141DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 142EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 143EEELQIIQPDKSVLVAAGETATLRCTITSLEPVGPIQWERGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVEINAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 144QVQLKQSGPGLVQPSQSLSITCTVSGESLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCRKTHTCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK 145EEELQIIQPDKSVLVAAGETATLRCTITSLEPVGPIQWERGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSEKTHTCPECPAPEAAGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 146EEELQVIQPDKSVLVAAGETATLRCTATSLEPVGPIQWERGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 147DKTHTCPPCPAPEAAGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 148EEELQVIQPDKSVLVAAGETATLRCTATSLEPVGPIQWERGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 149DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In addition to the knob-into-hole strategy, in some embodiments,electrostatic steering is also used to control the dimerization of Fcdomains. Electrostatic steering refers to the utilization of favorableelectrostatic interactions between oppositely charged amino acids inpeptides, protein domains, and proteins to control the formation ofhigher ordered protein molecules. In particular, to control thedimerization of Fc domains using electrostatic steering, one or moreamino acid residues that make up the CH3-CH3 interface are replaced withpositively- or negatively-charged amino acid residues such that theinteraction becomes electrostatically favorable or unfavorable dependingon the specific charged amino acids introduced. In some embodiments, apositively-charged amino acid in the interface, such as lysine,arginine, or histidine, is replaced with a negatively-charged amino acidsuch as aspartic acid or glutamic acid. In some embodiments, anegatively-charged amino acid in the interface is replaced with apositively-charged amino acid. In some embodiments, the charged aminoacids are introduced to one of the interacting CH3 antibody constantdomains, or both. In some embodiments, introducing charged amino acidsto the interacting CH3 antibody constant domains of the two Fc domainspromotes the selective formation of heterodimers of Fc domains ascontrolled by the electrostatic steering effects resulting from theinteraction between charged amino acids. Examples of electrostaticsteering amino acid pairs are included, without limitation, in Table 11.

TABLE 11 Electrostatic Steering Amino Acid Pairs Fc domain K409D K409DK409E K409E K392D K392D K392E K392E K409D K370E monomer 1 K392D K409DK439E Fc domain D399K D399R D399K D399R D399K D399R D399K D399R D399KD356K monomer 2 D356K E357K D399K

Other methods used to control the heterodimerization of Fc domains,especially in the context of constructing a bispecific antibody, areavailable.

In some embodiments, a first Fc domain and a second Fc domain eachincludes one or more of the following amino acid substitutions: T366W,T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T,L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R,E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E,K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N,F405T, F405H, F405R, Y407T, Y407H, Y4071, K409E, K409D, K409T, andK4091, relative to the sequence of human IgG1.

In some embodiments an Fc domain comprises: (a) one of the followingamino acid substitutions relative to wild type human IgG1: T366W, T366S,L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H,L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q,S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D,T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T,F405H, F405R, Y407T, Y407H, Y4071, K409E, K409D, K409T, or K4091; or (b)(i) a N297A mutation relative to a human IgG1 Fc region; (ii) a L234A,L235A, and G237A mutation relative to a human IgG1 Fc region; (iii) aL234A, L235A, G237A, and N297A mutation relative to a human IgG1 Fcregion; (iv) a N297A mutation relative to a human IgG2 Fc region; (v) aA330S and P331S mutation relative to a human IgG2 Fc region; (vi) aA330S, P331S, and N297A mutation relative to a human IgG2 Fc region;(vii) a S228P, E233P, F234V, L235A, and delG236 mutation relative to ahuman IgG4 Fc region; or (viii) a S228P, E233P, F234V, L235A, delG236,and N297A mutation relative to a human IgG4 Fc region. In someembodiments an Fc domain variant comprises: (a) one of the followingamino acid substitutions relative to wild type human IgG1: T366W, T366S,L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H,L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q,S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D,T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T,F405H, F405R, Y407T, Y407H, Y4071, K409E, K409D, K409T, or K4091; and(b) further comprises (i) a N297A mutation relative to a human IgG1 Fcregion; (ii) a L234A, L235A, and G237A mutation relative to a human IgG1Fc region; (iii) a L234A, L235A, G237A, and N297A mutation relative to ahuman IgG1 Fc region; (iv) a N297A mutation relative to a human IgG2 Fcregion; (v) a A330S and P331S mutation relative to a human IgG2 Fcregion; (vi) a A330S, P331S, and N297A mutation relative to a human IgG2Fc region; (vii) a S228P, E233P, F234V, L235A, and delG236 mutationrelative to a human IgG4 Fc region; or (viii) a S228P, E233P, F234V,L235A, delG236, and N297A mutation relative to a human IgG4 Fc region.

In some embodiments, the first and second Fc domains include differentamino acid substitutions. In some embodiments, the first Fc domainincludes T366W. In some embodiments, the second Fc domain includesT366S, L368A, and Y407V. In some embodiments, the first Fc domainincludes D399K. In some embodiments, the second Fc domain includesK409D.

Linkers

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein α (SIRP-α) D1 variant comprising a SIRPα D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRPα D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRPα D1 domain at a residueselected from the group consisting of: residue 6, residue 27, residue31, residue 47, residue 53, residue 54, residue 56, residue 66, andresidue 92.

Also disclosed herein, in some embodiments, are polypeptides comprisingan Fc variant, wherein the Fc variant comprises an Fc domain dimercomprising two Fc domain variants, wherein each Fc domain variantindependently is selected from (i) a human IgG1 Fc region consisting ofmutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc regionconsisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fcregion comprising mutations S228P, E233P, F234V, L235A, delG236, andN297A.

In the present disclosure, a linker is used to describe a linkage orconnection between polypeptides or protein domains or associatednon-protein moieties. In some embodiments, a linker is a linkage orconnection between an Fc domain (or variant thereof) and a SIRPα D1domain variant. In some embodiments, the linker connects the C-terminusof the SIRPα D1 domain variant and the N-terminus of the Fc domainvariant, such that the two polypeptides are joined to each other intandem series.

In some embodiments, a linker is a simple covalent bond, e.g., a peptidebond, a synthetic polymer, or any kind of bond created from a chemicalreaction, e.g. chemical conjugation. When a linker is a peptide bond, insome embodiments, the carboxylic acid group at the C-terminus of oneprotein domain reacts with the amino group at the N-terminus of anotherprotein domain in a condensation reaction to form a peptide bond. Insome embodiments, the peptide bond is formed from synthetic meansthrough a conventional organic chemistry reaction, or by naturalproduction from a host cell, wherein a nucleic acid molecule encodingthe DNA sequences of both proteins (e.g., an Fc domain variant and aSIRPα D1 domain variant) in tandem series can be directly transcribedand translated into a contiguous polypeptide encoding both proteins bythe necessary molecular machineries (e.g., DNA polymerase and ribosome)in the host cell.

When a linker is a synthetic polymer, in some embodiments, the polymeris functionalized with reactive chemical functional groups at each endto react with the terminal amino acids at the connecting ends of twoproteins.

When a linker (except peptide bond mentioned above) is made from achemical reaction, in some embodiments, chemical functional groups(e.g., amine, carboxylic acid, ester, azide, or other functionalgroups), are attached synthetically to the C-terminus of one protein andthe N-terminus of another protein, respectively. In some embodiments,the two functional groups then react through synthetic chemistry meansto form a chemical bond, thus connecting the two proteins together.

Spacers

In the present disclosure, in some embodiments, a linker between an Fcdomain monomer and a SIRPα D1 variant polypeptide of the disclosure, isan amino acid spacer including about 1-200 amino acids. Suitable peptidespacers include peptide linkers containing flexible amino acid residuessuch as glycine and serine. Examples of linker sequences are provided inTable 12. In some embodiments, a spacer contains motifs, e.g., multipleor repeating motifs, of GS, GG, GGS, GGG, GGGGS (SEQ ID NO: 163), GGSG(SEQ ID NO: 164), or SGGG (SEQ ID NO: 165). In some embodiments, aspacer contains 2 to 12 amino acids including motifs of GS, e.g., GS,GSGS (SEQ ID NO: 166), GSGSGS (SEQ ID NO: 167), GSGSGSGS (SEQ ID NO:168), GSGSGSGSGS (SEQ ID NO: 169), or GSGSGSGSGSGS (SEQ ID NO: 170). Insome embodiments, a spacer contains 3 to 12 amino acids including motifsof GGS, e.g., GGS, GGSGGS (SEQ ID NO: 171), GGSGGSGGS (SEQ ID NO: 172),and GGSGGSGGSGGS (SEQ ID NO: 173). In some embodiments, a spacercontains 4 to 12 amino acids including motifs of GGSG (SEQ ID NO: 164),e.g., GGSG (SEQ ID NO: 164), GGSGGGSG (SEQ ID NO: 174), or GGSGGGSGGGSG(SEQ ID NO: 175). In some embodiments, a spacer contains motifs of GGGGS(SEQ ID NO: 163), e.g., GGGGSGGGGSGGGGS (SEQ ID NO: 176). In someembodiments, a spacer contains amino acids other than glycine andserine, e.g., AAS (SEQ ID NO: 177), AAAL (SEQ ID NO: 178), AAAK (SEQ IDNO: 179), AAAR (SEQ ID NO: 180), EGKSSGSGSESKST (SEQ ID NO: 181),GSAGSAAGSGEF (SEQ ID NO: 182), AEAAAKEAAAKA (SEQ ID NO: 183),KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), GGGGAGGGG (SEQ ID NO: 185),GENLYFQSGG (SEQ ID NO: 186), SACYCELS (SEQ ID NO: 187), RSIAT (SEQ IDNO: 188), RPACKIPNDLKQKVIVINH (SEQ ID NO: 189),GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 190), AAANSSIDLISVPVDSR(SEQ ID NO: 191), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO:192).

In some embodiments, a spacer contains motifs, e.g., multiple orrepeating motifs, of EAAAK (SEQ ID NO: 193). In some embodiments, aspacer contains motifs, e.g., multiple or repeating motifs, ofproline-rich sequences such as (XP)n, in which X is any amino acid(e.g., A, K, or E) and n is from 1-5, and PAPAP (SEQ ID NO: 194).

TABLE 12 Linker Sequences SEQ ID NO: AMINO ACID SEQUENCE 163 GGGGS 164GGSG 165 SGGG 166 GSGS 167 GSGSGS 168 GSGSGSGS 169 GSGSGSGSGS 170GSGSGSGSGSGS 171 GGSGGS 172 GGSGGSGGS 173 GGSGGSGGSGGS 174 GGSGGGSG 175GGSGGGSGGGSG 176 GGGGSGGGGSGGGGS 177 AAS 178 AAAL 179 AAAK 180 AAAR 181EGKSSGSGSESKST 182 GSAGSAAGSGEF 183 AEAAAKEAAAKA 184 KESGSVSSEQLAQFRSLD185 GGGGAGGGG 186 GENLYFQSGG 187 SACYCELS 188 RSIAT 189RPACKIPNDLKQKVMNH 190 GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG 191AAANSSIDLISVPVDSR 192 GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS 193 EAAAK 194PAPAP

In some embodiments, the length of the peptide spacer and the aminoacids used is adjusted depending on the two proteins involved and thedegree of flexibility desired in the final protein fusion polypeptide.In some embodiments, the length of the spacer is adjusted to ensureproper protein folding and avoid aggregate formation. In someembodiments, a spacer is A or AAAL (SEQ ID NO: 178).

Vectors, Host Cells, and Protein Production

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein a (SIRP-α) D1 variant comprising a SIRPα D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRPα D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRPα D1 domain at a residueselected from the group consisting of: residue 6, residue 27, residue31, residue 47, residue 53, residue 54, residue 56, residue 66, andresidue 92.

Also disclosed herein, in some embodiments, are polypeptides comprisingan Fc variant, wherein the Fc variant comprises an Fc domain dimerhaving two Fc domain monomers, wherein each Fc domain monomerindependently is selected from (i) a human IgG1 Fc region consisting ofmutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc regionconsisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fcregion comprising mutations S228P, E233P, F234V, L235A, delG236, andN297A.

In some embodiments, the polypeptides of the disclosure are producedfrom a host cell. A host cell refers to a vehicle that includes thenecessary cellular components, e.g., organelles, needed to express thepolypeptides and fusion polypeptides described herein from theircorresponding nucleic acids. In some embodiments, the nucleic acids areincluded in nucleic acid vectors introduced into the host cell bytransformation, transfection, electroporation, calcium phosphateprecipitation, direct microinjection, infection, etc. In someembodiments, the choice of nucleic acid vector depends on the host cellto be used. In some embodiments, host cells are of either prokaryotic(e.g., bacterial) or eukaryotic (e.g., mammalian) origin.

In some embodiments, a polypeptide, for example a polypeptide constructcomprising a SIRPα D1 domain variant (e.g., any variant provided inTables 2, 5, and 6) and a fusion partner such as an Fc variant areproduced by culturing a host cell transformed with a nucleic acid,preferably an expression vector, containing a nucleic acid encoding thepolypeptide construct (e.g., Fc variant, linker, and fusion partner)under the appropriate conditions to induce or cause expression of thepolypeptide construct. In some embodiments, the conditions appropriatefor expression varies with the expression vector and the host cellchosen. In some embodiments, a wide variety of appropriate host cellsare used, including, but not limited to, mammalian cells, bacteria,insect cells, and yeast. For example, a variety of cell lines that finduse in the present disclosure are described in the ATCC® cell linecatalog, available from the American Type Culture Collection. In someembodiments, Fc domain variants of this disclosure are expressed in acell that is optimized not to glycosylate proteins that are expressed bysuch cell, either by genetic engineering of the cell line ormodifications of cell culture conditions such as addition of kifunensineor by using a naturally non-glycosylating host such as a prokaryote (E.coli, etc.), and in some cases, modification of the glycosylationsequence in the Fc is not be needed.

Nucleic Acid Vector Construction and Host Cells

A nucleic acid sequence encoding the amino acid sequence of apolypeptide of the disclosure can be prepared by a variety of methods.These methods include, but are not limited to, oligonucleotide-mediated(or site-directed) mutagenesis and PCR mutagenesis. In some embodiments,a nucleic acid molecule encoding a polypeptide of the disclosure isobtained using standard techniques, e.g., gene synthesis. Alternatively,a nucleic acid molecule encoding a wild-type SIRPα D1 domain is mutatedto include specific amino acid substitutions using standard techniques,e.g., QuikChange™ mutagenesis. In some cases, nucleic acid molecules aresynthesized using a nucleotide synthesizer or PCR techniques.

In some embodiments, the nucleic acids that encode a polypeptideconstruct, for example a polypeptide construct comprising a SIRPα D1domain variant (e.g., any variant provided in Tables 2, 5, and 6) and afusion partner such as an Fc variant are incorporated into an expressionvector in order to express the protein. A variety of expression vectorscan be utilized for protein expression. Expression vectors can compriseself-replicating, extra-chromosomal vectors or vectors which integrateinto a host genome. A vector can also include various components orelements. For example, in some embodiments, the vector componentsinclude, but are not limited to, transcriptional and translationalregulatory sequences such as a promoter sequence, a ribosomal bindingsite, a signal sequence, transcriptional start and stop sequences,translational start and stop sequences, 3′ and 5′ untranslated regions(UTRs), and enhancer or activator sequences; an origin of replication; aselection marker gene; and the nucleic acid sequence encoding thepolypeptide of interest, and a transcription termination sequence. Insome embodiments, expression vectors comprise a protein operably linkedwith control or regulatory sequences, selectable markers, any fusionpartners, additional elements, or any combinations thereof. The term“operably linked” means that the nucleic acid is placed into afunctional relationship with another nucleic acid sequence. Generally,these expression vectors include transcriptional and translationalregulatory nucleic acid operably linked to the nucleic acid encoding theFc variant, and are typically appropriate to the host cell used toexpress the protein. A selection gene or marker, such as, but notlimited to, an antibiotic resistance gene or fluorescent protein gene,can be used to select for host cells containing the expression vector,for example by antibiotic or fluorescence expression. Various selectiongenes are available.

In some embodiments, the components or elements of a vector areoptimized such that expression vectors are compatible with the host celltype. Expression vectors which find use in the present disclosureinclude, but are not limited to, those which enable protein expressionin mammalian cells, bacteria, insect cells, yeast, and in in vitrosystems.

In some embodiments, mammalian cells are used as host cells to producepolypeptides of the disclosure. Examples of mammalian cell typesinclude, but are not limited to, human embryonic kidney (HEK) (e.g.,HEK293, HEK 293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero,MC3T3, NS0, Sp2/0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20,T47D, NS0 (a murine myeloma cell line that does not endogenously produceany immunoglobulin chains), CRL7O3O, and HsS78Bst cells. In someembodiments, E. coli cells are used as host cells to producepolypeptides of the disclosure. Examples of E. coli strains include, butare not limited to, E. coli 294 (ATCC® 31,446), E. coli λ 1776 (ATCC®31,537, E. coli BL21 (DE3) (ATCC® BAA-1025), and E. coli RV308 (ATCC®31,608).

Different host cells have characteristic and specific mechanisms for theposttranslational processing and modification of protein products (e.g.,glycosylation). In some embodiments, appropriate cell lines or hostsystems are chosen to ensure the correct modification and processing ofthe polypeptide expressed. Once the vectors are introduced into hostcells for protein production, host cells are cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

In some embodiments, a polypeptide construct, for example a polypeptideconstruct comprising a SIRPα D1 domain variant (e.g., any variantprovided in Tables 2, 5, and 6) and a fusion partner such as an Fcvariant are expressed in mammalian expression systems, including systemsin which the expression constructs are introduced into the mammaliancells using virus such as retrovirus or adenovirus. In some embodiments,human, mouse, rat, hamster, or primate cells are utilized. Suitablecells also include known research cells, including but not limited toJurkat T cells, NIH3T3, CHO, COS, and 293 cells. Alternately, in someembodiments, proteins are expressed in bacterial cells. Bacterialexpression systems are well known in the art, and include Escherichiacoli (E. coli), Bacillus subtilis, Streptococcus cremoris, andStreptococcus lividans. In some cases, polypeptide constructs comprisingFc domain variants are produced in insect cells such as but not limitedto Sf9 and Sf21 cells or yeast cells such as but not limited toorganisms from the genera Saccharomyces, Pichia, Kluyveromyces,Hansenula and Yarrowia. In some cases, polypeptide constructs comprisingFc domain variants are expressed in vitro using cell free translationsystems. In vitro translation systems derived from both prokaryotic(e.g., E. coli) and eukaryotic (e.g., wheat germ, rabbit reticulocytes)cells are available and, in some embodiments, chosen based on theexpression levels and functional properties of the protein of interest.For example, as appreciated by those skilled in the art, in vitrotranslation is required for some display technologies, for exampleribosome display. In addition, in some embodiments, the Fc domainvariants are produced by chemical synthesis methods such as, but notlimited to, liquid-phase peptide synthesis and solid-phase peptidesynthesis. In the case of in vitro transcription using anon-glycosylating system such as bacterial extracts, the Fc will not beglycosylated even in presence of the natural glycosylation site andtherefore inactivation of the Fc will be equivalently obtained.

In some embodiments, a polypeptide construct includes non-natural aminoacids, amino acid analogues, amino acid mimetics, or any combinationsthereof that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids generally refer to the 20common amino acids (alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine) and pyrrolysine and selenocysteine.Amino acid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, e.g., a carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,such as, homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. In some embodiments, such analogs have modified R groups(such as, norleucine) or modified peptide backbones, but generallyretain the same basic chemical structure as a naturally occurring aminoacid.

Protein Production, Recovery, and Purification

In some embodiments, host cells used to produce polypeptides of thedisclosure are grown in media suitable for culturing of the selectedhost cells. Examples of suitable media for mammalian host cells includeMinimal Essential Medium (MEM), Dulbecco's Modified Eagle's Medium(DMEM), Expi293™ Expression Medium, DMEM with supplemented fetal bovineserum (FBS), and RPMI-1640. Examples of suitable media for bacterialhost cells include Luria broth (LB) plus necessary supplements, such asa selection agent, e.g., ampicillin. In some embodiments, host cells arecultured at suitable temperatures, such as from about 20° C. to about39° C., e.g., from about 25° C. to about 37° C., preferably 37° C., andCO₂ levels, such as about 5% to 10%. In some embodiments, the pH of themedium is from about pH 6.8 to pH 7.4, e.g., pH 7.0, depending mainly onthe host organism. If an inducible promoter is used in the expressionvector, protein expression can be induced under conditions suitable forthe activation of the promoter.

In some embodiments, protein recovery involves disrupting the host cell,for example by osmotic shock, sonication, or lysis. Once the cells aredisrupted, cell debris is removed by centrifugation or filtration. Theproteins can then be further purified. In some embodiments, apolypeptide of the disclosure is purified by various methods of proteinpurification, for example, by chromatography (e.g., ion exchangechromatography, affinity chromatography, and size-exclusion columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. For example,in some embodiments, the protein is isolated and purified byappropriately selecting and combining affinity columns such as Protein Acolumn (e.g., POROS Protein A chromatography) with chromatographycolumns (e.g., POROS HS-50 cation exchange chromatography), filtration,ultra-filtration, de-salting and dialysis procedures. In someembodiments, a polypeptide is conjugated to marker sequences, such as apeptide to facilitate purification. An example of a marker amino acidsequence is a hexa-histidine peptide (His6-tag (SEQ ID NO: 223)), whichcan bind to a nickel-functionalized agarose affinity column withmicromolar affinity. As an alternative, a hemagglutinin “HA” tag, whichcorresponds to an epitope derived from the influenza hemagglutininprotein can be used.

In some embodiments, polypeptides of the disclosure, for example apolypeptide construct comprising a SIRPα D1 domain variant (e.g., anyvariant provided in Tables 2, 5, and 6) and a fusion partner such as anFc variant are produced by the cells of a subject (e.g., a human), e.g.,in the context of gene therapy, by administrating a vector such as aviral vector (e.g., a retroviral vector, adenoviral vector, poxviralvector (e.g., vaccinia viral vector, such as Modified Vaccinia Ankara(MVA)), adeno-associated viral vector, and alphaviral vector) containinga nucleic acid molecule encoding a polypeptide of the disclosure. Thevector, once inside a cell of the subject (e.g., by transformation,transfection, electroporation, calcium phosphate precipitation, directmicroinjection, infection, etc.) can be used for the expression of apolypeptide disclosed herein. In some cases, the polypeptide is secretedfrom the cell. In some embodiments, if treatment of a disease ordisorder is the desired outcome, no further action is required. In someembodiments, if collection of the protein is desired, blood is collectedfrom the subject and the protein purified from the blood by variousmethods.

Methods of Treating Cancer

Provided herein are methods of treating cancer in an individual (e.g., ahuman individual) that comprises administering to the individual aneffective amount of (a) an agent that blocks the interaction betweenCD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) and (b) a chemotherapy agent(e.g., at least one chemotherapy agent, such as at least two, at leastthree, or at least four chemotherapy agents). Provided herein is amethod of treating cancer in an individual (e.g., a human individual)that comprises administering to the individual an effective amount of(a) a polypeptide comprising a SIRPα D1 domain variant (e.g., a SIRPα D1domain variant described herein) and an Fc domain variant (e.g., an Fcdomain variant described herein) and (b) a chemotherapy agent (e.g., atleast one chemotherapy agent, such as at least two, at least three, orat least four chemotherapy agents). In some embodiments the methodfurther comprises administering to the individual an effective amount ofa therapeutic antibody (e.g., at least one therapeutic antibody, such asat least two, at least three, or at least four therapeutic antibodies).Additionally or alternatively, in some embodiments the method furthercomprises administering to the individual an effective amount of animmunotherapeutic agent (e.g., at least one immunotherapeutic agent,such as at least two, at least three, or at least four immunotherapeuticagents). Additionally or alternatively, in some embodiments, the methodcomprises administering the polypeptide and the chemotherapy agent incombination with one or more additional modes of therapy, including, butnot limited to, e.g., radiation therapy, surgery, cryoablation, and bonemarrow transplant.

Combination Therapies Comprising Chemotherapy Agents, and ExemplaryChemotherapy Agents

Exemplary chemotherapy agent(s) that can be used in a method of treatingcancer described herein include, without limitation, e.g., methotrexate(RHEUMATREX®, Amethopterin), cyclophosphamide (CYTOXAN®), abiraterone,abemaciclib, altretamine, thalidomide (THALIDOMID®), acridinecarboxamide, Actimid®, actinomycin, actinomycin-D, afatinib,17-N-allylamino-17-demethoxygeldanamycin, alectinib, alpelisib,aminopterin, amsacrine, anlotinib, anthracycline, antineoplastic,antineoplaston, apartinib, 5-azacitidine, 6-mercaptopurine,6-thioguanine, arabinosylcytosine, axitinib, azacitidine, azathioprine,BL22, bendamustine, binimetinib, biricodar, bleomycin, bortezomib,bosutinib, brigatinib, bryostatin, busulfan, cabozantinib, calyculin,camptothecin, capecitabine, carboplatin, carmustine, ceritinib,chlorambucil, cisplatin, cladribine, clofarabine, cobimetinib,crizotinib, cytarabine, dabrafenib, dacarbazine, dacomitinib, dasatinib,daunorubicin, dexamethasone, dichloroacetic acid, discodermolide,docetaxel, doxorubicin, encorafenib, epirubicin, entrectinib,enzalutamide, epothilone, erdafitinib, eribulin, erlotinib,estramustine, etoposide, everolimus, exatecan, exisulind, ferruginol,floxuridine, fludarabine, fluorouracil (such as 5-fluorouracil), folinicacid, fosfestrol, fotemustine, fruquintinib, ganciclovir, gefitinib,gemcitabine, gilteritinib, goserelin, hexamethylmelamine,hydroxycarbamide, hydroxyurea, IT-101, ibrutinib, icotinib, idarubicin,idelalisib, ifosfamide, imatinib, irinoimiquimod, irinotecan, irofulven,ivosidenib, ixabepilone, laniquidar, lapatinib, larotrectinib,lenalidomide, lenvatinib, lorlatinib, lomustine, lurtotecan,mafosfamide, masoprocol, mechlorethamine, melphalan, mercaptopurine,methotrexate, methylprednisolone, mitomycin, mitotane, mitoxantrone,nelarabine, neratinib, niraparib, nilotinib, nintedanib, oblimersen,olaparib, osimertinib, oxaliplatin, nedaplatin, phenanthriplatin,picoplatin, PAC-1, paclitaxel, palbociclib, pazopanib, pemetrexed,pegfilgrastim, pentostatin, pipobroman, pixantrone, plicamycin,prednisone, ponatinib, procarbazine, proteasome inhibitors (e.g.,bortezomib), pyrotinib, raltitrexed, rebeccamycin, Revlimid®,regorafenib, ribociclib, rubitecan, rucaparib, ruxolitinib, SN-38,salinosporamide A, satraplatin, sirolimus, sonidegib, sorafenib,streptozocin, streptozotocin, sunitinib, swainsonine, talazoparib,tariquidar, taxane, tegafur-uracil, temsirolimus, teniposide,temozolomide, testolactone, thioTEPA, tioguanine, topotecan,trabectedin, trametinib, tretinoin, trifluridine, triplatintetranitrate, tris(2-chloroethyl)amine, troxacitabine, uracil mustard,valrubicin, vandetanib, vemurafenib, venetoclax (ABT-199), navitoclax(ABT-263), vinblastine, vincristine, vinorelbine, vismodegib,vorinostat, ziv-aflibercept (ZALTRAP®), zosuquidar, or the like.

In some embodiments, the method of treating cancer comprisesadministering an agent that blocks the interaction between CD47 (e.g.,hCD47) and SIRPα (e.g., hSIRPα) in combination with chemotherapeuticagent(s)s of a particular class. In some embodiments, the agent thatblocks the interaction between CD47 and SIRPα is a polypeptide describedherein (e.g., a fusion polypeptide comprising a SIRPα d1 domain variantand an Fc variant; a fusion polypeptide comprising a SIRPγ variant, aSIRPβ1 variant, or a SIRPβ2 variant and an Fc variant). For example, insome embodiments, the method of treating cancer comprises administeringa polypeptide (e.g. fusion polypeptide) described herein in combinationwith an adrenal inhibitor (including, but not limited to adrenalinhibitors described herein). For example, in some embodiments, themethod of treating cancer comprises administering a polypeptidedescribed herein in combination with an anthracycline (including, butnot limited to anthracyclines described herein). In some embodiments,the method of treating cancer comprises administering a polypeptidedescribed herein in combination with an alkylating agent (including, butnot limited to alkylating agents described herein). In some embodiments,the method of treating cancer comprises administering a polypeptidedescribed herein in combination with an androgen inhibitor (including,but not limited to androgen inhibitors described herein). In someembodiments, the method of treating cancer comprises administering apolypeptide described herein in combination with an antimetabolite,e.g., a purine analog, (including, but not limited to antimetabolites,e.g., purine analogs, described herein). In some embodiments, the methodof treating cancer comprises administering a polypeptide describedherein in combination with an antitumor antibiotic (including, but notlimited to antitumor antibiotics described herein. In some embodiments,the method of treating cancer comprises administering a polypeptidedescribed herein in combination with a BLC-2 inhibitor (including, butnot limited to BLC-2 inhibitors described herein). In some embodiments,the method of treating cancer comprises administering a polypeptidedescribed herein in combination with a BTK inhibitor (including, but notlimited to BTK inhibitors described herein. In some embodiments, themethod of treating cancer comprises administering a polypeptidedescribed herein in combination with a CDK 4/6 inhibitor (including, butnot limited to CDK 4/6 inhibitors described herein). In someembodiments, the method of treating cancer comprises administering apolypeptide described herein in combination with a colony stimulatingfactor (including, but not limited to colony stimulating factorsdescribed herein). In some embodiments, the method of treating cancercomprises administering a polypeptide described herein in combinationwith a corticosteroid (including, but not limited to corticosteroidsdescribed herein). In some embodiments, the method of treating cancercomprises administering a polypeptide described herein in combinationwith an EGFR inhibitor (including, but not limited to EGFR inhibitorsdescribed herein). In some embodiments, the method of treating cancercomprises administering a polypeptide described herein in combinationwith a gonadotropin releasing hormone (GnRH) agonist (including, but notlimited to GnRH agonists described herein). In some embodiments, themethod of treating cancer comprises administering a polypeptidedescribed herein in combination with a mitotic inhibitor/microtubuleinhibitor (including, but not limited to mitotic inhibitors/microtubuleinhibitors described herein). In some embodiments, the method oftreating cancer comprises administering a polypeptide described hereinin combination with an mTOR kinase inhibitor (including, but not limitedto mTOR kinase inhibitors described herein). In some embodiments, themethod of treating cancer comprises administering a polypeptidedescribed herein in combination with a proteasome inhibitor (including,but not limited to proteasome inhibitors described herein). In someembodiments, the method of treating cancer comprises administering apolypeptide described herein in combination with a signal transductioninhibitor, e.g., a protein-tyrosine kinase inhibitor, a PAK4 inhibitor,a PI3K inhibitor, (including, but not limited to signal transductioninhibitors described herein). In some embodiments, the method oftreating cancer comprises administering a polypeptide described hereinin combination with a topoisomerase inhibitor, (including, but notlimited to topoisomerase inhibitors described herein). In someembodiments, the method of treating cancer comprises administering apolypeptide described herein in combination with a tyrosine kinaseinhibitor, (including, but not limited to tyrosine kinase inhibitorsdescribed herein). In some embodiments, the method of treating cancercomprises administering a polypeptide described herein in combinationwith a VEGF inhibitor, such as a VEGF1 inhibitor, a VEGF2 inhibitor,and/or a VEGF3 inhibitor (including, but not limited to VEGF inhibitorsdescribed herein. In some embodiments, the method of treating cancercomprises administering a polypeptide described herein in combinationwith an agent that modulates apoptosis, e.g., by modulating the activityof Bcl-2, Mcl1, Bcl-lx, etc., (including, but not limited to agents thatmodulate apoptosis, e.g., by modulating the activity of Bcl-2, Mcl1,Bcl-lx, etc., described herein). In some embodiments, the method oftreating cancer comprises administering a polypeptide described hereinin combination with a platinum-based agent, (including, but not limitedto platinum-based agents described herein). In some embodiments, themethod of treating cancer comprises administering a polypeptidedescribed herein in combination with an inhibitor of NTRK1, NTRK2,and/or NTRK3, an ALK inhibitor, a ROS inhibitor, a FLT3 inhibitor, aBRAF inhibitor, an inhibitor of MEK1 and/or MEK2, an inhibitor of HER2,HER3, and/or HER 4, an inhibitor of RET/PTC, an inhibitor of BCR-ABL, ac-KIT inhibitor, an inhibitor of PDGFR-alpha and/or PDGFR-beta, aninhibitor of FGFR1, FGFR2, FGFR3, and/or FGFR4, an Smoothened inhibitorand/or an inhibitor of PARP1, PARP2, and/or PARP3 (including, but notlimited to inhibitors described herein). In some embodiments, theinhibitor is an antisense polynucleotide (such as an siRNA or an RNAi).In some embodiments, the inhibitor is a small molecule inhibitor, asdescribed in further detail below.

In some embodiments the chemotherapeutic agent is a small moleculeanti-cancer agent (such as a small molecule inhibitor In someembodiments, the method of treating cancer comprises administering apolypeptide described herein in combination with a small moleculeinhibitor of VEGFR and/or PDGFR, a small molecule EGFR inhibitor, asmall molecule ALK inhibitor, a small molecule CDK4/6 inhibitor, a smallmolecule PARP inhibitor, a small molecule PAK4 inhibitor, a smallmolecule mTOR inhibitor, a small molecule KRAS inhibitor, a smallmolecule TRK inhibitor, a small molecule BCL2 inhibitor, a smallmolecule B-raf inhibitor, a small molecule IDH inhibitor, a smallmolecule PI3K inhibitor, a small molecule DDR (DNA damage response)inhibitor, or a small molecule hypomethylation agent. In other cases,the targeted small molecule modulates a cellular signaling pathway ofthe cell expressing CD47, e.g., an IDO/TDO inhibitor, AhR inhibitor,arginase inhibitor, A2a R inhibitor, TLR agonists, STING agonist, orRig-1 agonist.

In some embodiments, the method of treating cancer comprisesadministering a polypeptide described herein (e.g., a fusion polypeptidecomprising a SIRPα d1 domain variant and an Fc variant) in combinationwith at least one, at least two, at least three, or at least fourchemotherapeutic agents. In some embodiments where two or morechemotherapeutic agents are administered, the two or morechemotherapeutic agents are from different classes (as described above)and/or exert their anti-cancer effects via different mechanisms ofaction.

Further details regarding exemplary pharmaceutical compositions andpreparations, exemplary dosages, and exemplary routes of administrationfor the fusion polypeptides described herein are provided in WO2017/027422 and U.S. Pat. No. 10,259,859, the contents of each of whichare incorporated by reference entireties.

Combination Therapies Comprising Therapeutic Antibodies, and ExemplaryTherapeutic Antibodies

In some embodiments a method of treating cancer provided hereincomprises administering to the individual an effective amount of atherapeutic antibody (e.g., at least one therapeutic antibody, such asat least two, at least three, or at least four therapeutic antibodies),i.e., in combination with agent that blocks the interaction between CD47(e.g., hCD47) and SIRPα (e.g., a fusion polypeptide described herein)and a chemotherapeutic agent described herein (e.g., at least onechemotherapeutic agent, such as at least two, at least three, or atleast four chemotherapeutic agents). In some embodiments, thetherapeutic antibody is conjugated to a drug (i.e., an antibody-drugconjugate, or “ADC”).

Exemplary therapeutic antibodies (e.g., therapeutic monoclonalantibodies) for use in a method herein include, but are not limited to,e.g., 3F8, 8H9, Abagovomab, Abciximab, Abituzumab, Abrilumab, Actoxumab,Adalimumab, Adecatumumab, Aducanumab, Afelimomab, Afutuzumab, Alacizumabpegol, ALD518, Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab,Anatumomab mafenatox, Anetumab ravtansine, Anifrolumab, Anrukinzumab(IMA-638), Apolizumab, Arcitumomab, Ascrinvacumab, Aselizumab,Atezolizumab, Atinumab, Atlizumab (tocilizumab), Atorolimumab, Avelumab,Bapineuzumab, Basiliximab, Bavituximab, Bectumomab, Begelomab,Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab,Bezlotoxumab, Biciromab, Bimagrumab, Bimekizumab, Bivatuzumabmertansine, Blinatumomab, Blosozumab, Bococizumab, Brentuximab vedotin,Briakinumab, Brodalumab, Brolucizumab, Brontictuzumab, Cabiralizumab(FPA008), Camrelizumab, Canakinumab, Cantuzumab mertansine, Cantuzumabravtansine, Caplacizumab, Capromab pendetide, Carlumab, Catumaxomab,cBR96-doxorubicin immunoconjugate, CC49, Cedelizumab, Certolizumabpegol, Cetuximab, Ch.14.18, Citatuzumab bogatox, Cixutumumab,Clazakizumab, Clenoliximab, Clivatuzumab tetraxetan, Codrituzumab,Coltuximab ravtansine, Conatumumab, Concizumab, Crenezumab, CR6261,Dacetuzumab, Daclizumab, Dalotuzumab, Dapirolizumab pegol, Daratumumab,Dectrekumab, Demcizumab, Denintuzumab mafodotin, Denosumab, Derlotuximabbiotin, Detumomab, Dinutuximab, Diridavumab, Dorlimomab aritox,Drozitumab, Duligotumab, Dupilumab, Durvalumab, Dusigitumab,Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab,Eldelumab, Elgemtumab, Elotuzumab, Elsilimomab, Emactuzumab (RG7155),Emibetuzumab, Enavatuzumab, Enfortumab vedotin, Enlimomab pegol,Enoblituzumab, Enokizumab, Enoticumab, Ensituximab, Epitumomabcituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab,Etrolizumab, Evinacumab, Evolocumab, Exbivirumab, Fanolesomab,Faralimomab, Farletuzumab, Fasinumab, FBTA05, Felvizumab, Fezakinumab,Ficlatuzumab, Figitumumab, Firivumab, Flanvotumab, Fletikumab,Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulranumab,Futuximab, Galiximab, Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumabozogamicin, Gevokizumab, Girentuximab, Glembatumumab vedotin, Golimumab,Gomiliximab, Guselkumab, Ibalizumab, Ibritumomab tiuxetan, Icrucumab,Idarucizumab, Igovomab, IMAB362, Imalumab, Imciromab, Imgatuzumab,Inclacumab, Indatuximab ravtansine, Indusatumab vedotin, Infliximab,Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab,Isatuximab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab,Lambrolizumab, Lampalizumab, Lebrikizumab, Lemalesomab, Lenzilumab,Lerdelimumab, Lexatumumab, Libivirumab, Lifastuzumab vedotin,Ligelizumab, Lilotomab satetraxetan, Lintuzumab, Lirilumab,Lodelcizumab, Lokivetmab, Lorvotuzumab mertansine, Lucatumumab,Lulizumab pegol, Lumiliximab, Lumretuzumab, MSB0010718C (avelumab),Mapatumumab, Margetuximab, Maslimomab, Mavrilimumab, Matuzumab,MEDI6469, MEDI0680, MEDI6383, Mepolizumab, Metelimumab, Milatuzumab,Minretumomab, Mitumomab, Mogamulizumab, Morolimumab, Motavizumab,Moxetumomab pasudotox, Muromonab-CD3, Nacolomab tafenatox, Namilumab,Naptumomab estafenatox, Narnatumab, Natalizumab, Nebacumab, Necitumumab,Nemolizumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab,Nofetumomab merpentan, Obiltoxaximab, Obinutuzumab, Ocaratuzumab,Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab,Onartuzumab, Ontuxizumab, Opicinumab, Oportuzumab monatox, Oregovomab,Orticumab, Otelixizumab, Otlertuzumab, Oxelumab, Ozanezumab,Ozoralizumab, Pagibaximab, Palivizumab, Panitumumab, Pankomab,Panobacumab, Parsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab,Patritumab, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab,Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab,Polatuzumab vedotin, Ponezumab, Priliximab, Pritoxaximab, Pritumumab,PRO 140, Quilizumab, Racotumomab, Radretumab, Rafivirumab,Ralpancizumab, Ramucirumab, Ranibizumab, Raxibacumab, Refanezumab,Regavirumab, Reslizumab, Rilotumumab, Rinucumab, Rituximab, Robatumumab,Roledumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab,Sacituzumab govitecan, Samalizumab, SAR650984 (Isatuximab) Sarilumab,Satumomab pendetide, Secukinumab, Seribantumab, Setoxaximab, Sevirumab,Sibrotuzumab, SGN-CD19A, SGN-CD33A, Sifalimumab, Siltuximab, Simtuzumab,Sintilimab, Siplizumab, Sirukumab, Sofituzumab vedotin, Solanezumab,Solitomab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Suvizumab,Tabalumab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tanezumab,Taplitumomab paptox, Tarextumab, Tefibazumab, Telimomab aritox,Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, Tesidolumab,TGN1412, Ticilimumab (tremelimumab), Tildrakizumab, Tigatuzumab,TNX-650, Tocilizumab (atlizumab), Toralizumab, Toripalimab, Tosatoxumab,Tositumomab, Tovetumab, Tralokinumab, Trastuzumab,trastuzumab-emtansine, TRBS07, Tregalizumab, Tremelimumab, Tucotuzumabcelmoleukin, Tuvirumab, Ublituximab, Ulocuplumab, Urelumab, Urtoxazumab,Ustekinumab, Utomilumab (PF-05082566), Vandortuzumab vedotin,Vantictumab, Vanucizumab, Vapaliximab, Varlilumab, Vatelizumab,Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab,Volociximab, Vonlerolizumab (RG7888), Vorsetuzumab mafodotin, Votumumab,Zalutumumab, Zanolimumab, Zatuximab, Ziralimumab, or Zolimomab aritox,including biosimilars of any of the preceding therapeutic antibodies.

Other exemplary therapeutic antibodies (e.g., therapeutic monoclonalantibodies) that can be used in a method herein is an antibody include,but are not limited to, e.g., an anti-CD20 antibody, an anti-EGFRantibody, an anti-Her2/Neu (ERBB2) antibody, an anti-EPCAM antibody, ananti-GL2 antibody, anti-GD2, anti-GD3, anti-CD2, anti-CD3, anti-CD4,anti-CD8, anti-CD I 9, anti-CD22, anti-CD30, anti-CD33, anti-CD39,anti-CD45, anti-CD47, anti-CD52, anti-CD56, anti-CD70, anti-CD73,anti-CD117, an anti-SIRPα antibody, an anti-LILRB1, an anti-LILRB2, ananti-LILRB4 antibody, an anti-PD-1 antibody (e.g., an anti PD-1antagonist antibody), an anti-PD-L1 antibody (e.g., an anti PD-L1antagonist antibody), an anti-PD-L2 antibody, or any antibody designedto bind to a tumor cell, a virally- or bacterially-infected cell, immunecell, or healthy normal cell, or to a cytokine, chemokine, or hormone ofany kind.

In some embodiments, the therapeutic antibody used in a method herein isan antibody that binds to, e.g., CS1/SLAMF7, Trop-2, VWF, vimentin,VEGFR2, VEGFR-1, VEGF, VEGF-A, TYRP1 (glycoprotein 75), TWEAK receptor,tumor specific glycosylation of MUC1, tumor antigen CTAA16.88, TRAIL-R2,TRAIL-R1, TNF-alpha, TGF-beta, TGF beta 2, TGF beta 1, TFPI, tenascin C,TEM1, TAG-72, T-cell receptor, STEAP1, sphingosine-1-phosphate, SOST,SLAMF7, BCL-2, selectin P, SDC1, sclerostin, RTN4, RON, Rhesus factor,RHD, respiratory syncytial virus, RANKL, rabies virus glycoprotein,platelet-derived growth factor receptor beta, phosphatidylserine,phosphate-sodium co-transporter, PDGF-R alpha, PDCD1, PD-1, PD-L1,PCSK9, oxLDL, OX-40, NRP1, Notch receptor 4, Notch receptor 3, Notchreceptor 2, Notch receptor 1, NOGO-A, NGF, neural apoptosis-regulatedproteinase 1, NCA-90 (granulocyte antigen), NARP-1, N-glycolylneuraminicacid, myostatin, myelin-associated glycoprotein, mucin CanAg, MUC1,MSLN, MS4A1, MIF, mesothelin, MCP-1, LTA, LOXL2, lipoteichoic acid,LINGO-1, LFA-1 (CD11a), Lewis-Y antigen, L-selectin (CD62L), KIR2D,ITGB2 (CD18), ITGA2, interferon alpha/beta receptor, interferonreceptor, interferon gamma-induced protein, integrin αvβ3, integrinaIIβ3, integrin α7β7, integrin and, integrin α4β7, integrin α4,insulin-like growth factor I receptor, Influenza A hemagglutinin, ILGF2,IL9, IL6, IL4, IL3 IRA, IL23, ILI 7A, IL-6 receptor, IL-6, IL-S, IL-4,IL-23, IL-22, IL-I, IL-I 7A, IL-I 7, IL-13, IL-I 2, IL-I, IL 20, IGHE,IgG4, IGF-I, IGF-I receptor, IgE Fc region, IFN-gamma, IFN-alpha, ICAM-1(CD54), human TNF, human scatter factor receptor kinase, Hsp90, HNGF,HLA-DR, HIV-1, histone complex, HHGFR, HGF, HERS, HER2, HER2/neu, HER′,hepatitis B surface antigen, hemagglutinin, GUCY2C, GPNMB, GMCSFreceptor alpha-chain, glypican 3, GD3 ganglioside, GD2, ganglioside GD2,Frizzled receptor, folate receptor 1, folate hydrolase, fibronectinextra domain-B, fibrin II, beta chain, FAP, F protein of respiratorysyncytial virus, ERBB3, episialin, EpCAM, endotoxin, EGFR, EGFL7, E.coli shiga toxin type-2, E. coli shiga toxin type-I, DRS, DPP4, DLL4,dabigatran, cytomegalovirus glycoprotein B, CTLA-4, CSF2, CSF1R,clumping factor A, CLDN18.2, ch4DS, CFD, CEA-related antigen, CEA, CD80,CD79B, CD74, CD73, CD70, CD6, CD56, CD52, CD51, CD5, CD44 v6, CD41, CD40ligand, CD40, CD4, CD39, CD38, CD37, CD33, CD30 (TNFRSF8), CD123, CD138,CD3 epsilon, CD3, CD28, CD274, CD27, CD2S (a chain of IL-2 receptor),CD23 (IgE receptor), CD221, CD22, CD200, CD20, CD2, CD19, CD137, CD154,CD152, CD15, CD147 (basigin), CD140a, CD125, CD11, CD-18, CCR5, CCR4,CCL11 (eotaxin-I), cardiac myosin, carbonic anhydrase 9 (CA-IX), Canislupus familiaris IL31, CA-125, C5, C242 antigen, C-X-C chemokinereceptor type 4, beta-amyloid, BAFF, B7-H3, B-lymphoma cell, AOC3(VAP-I), anthrax toxin, protective antigen, angiopoietin 3, angiopoietin2, alpha-fetoprotein, AGS-22M6, adenocarcinoma antigen, ACVR2B, activinreceptor-like kinase I, 5T4, SAC, 4-IBB or 1-40-beta-amyloid.

In some embodiments, the therapeutic antibody used in a method hereinbinds to an antigen expressed by a cancer cell (e.g., expressed on thesurface of a cancer cell). Exemplary antigens expressed by cancers areknown in the art and include, without limitation, e.g., CD19, CD20,CD22, CD30, CD33, CD38, CD52, CD56, CD70, CD74, CD79b, CD123, CD138,CS1/SLAMF7, Trop-2, 5T4, BCMA, Mucin 1, Mucin 16, PTK7, PD-L1, STEAP1,Endothelin B Receptor, mesothelin, EGFRvIII, ENPP3, SLC44A4, GNMB,nectin 4, NaPi2b, LIV-1A, Guanylyl cyclase C, DLL3, EGFR, HER2, VEGF,VEGFR, integrin aVf33, integrin α501, MET, IGF1R, TRAILR1, TRAILR2,RANKL, FAP, Tenascin, Le″, EpCAM, CEA, gpA33, PSMA, TAG72, a mucin,CAIX, EPHA3, folate receptor a, GD2, GD3, and an MEC/peptide complexcomprising a peptide from NY-ESO-1/LAGE, SSX-2, a MAGE family protein,MAGE-A3, gp100/pme117, Melan-A/MART1, gp75/TRP1, tyrosinase, TRP2, CEA,PSA, TAG-72, immature laminin receptor, MOK/RAGE-1, WT-1, SAP-1, BING-4,EpCAM, MUC1, PRAME, survivin, BRCA1, BRCA2, CDK4, CML66, MART-2, p53,Ras, β-catenin, TGF-βRII, HPV E6, or HPV E7. For example, in someembodiments, an polypeptide described herein is administered incombination with a chemotherapeutic agent (e.g., at least onechemotherapeutic agent) and a monoclonal antibody that binds CD123 (alsoknown as IL-3 receptor alpha), such as talacotuzumab (also known asCSL362 and JNJ-56022473).

In some embodiments, the therapeutic antibody (e.g., therapeuticmonoclonal antibody) used in a method herein is an antibody that bindsan antigen expressed by an NK cell. Exemplary antigens expressed by anNK cell include, without limitation, NKR-P1A (KLRB1), CD94 (NKG2A),KLRG1, KIR2DL5A, KIR2DL5B, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DS2, KIR2DS3,KIR2DS4, KIR2DS5, KIR3DS1, KIR2DS1, CD94 (NKG2C/E), NKG2D, CD160 (BY55),CD16 (FcγRIIIA), NKp46 (NCR1), NKp30 (NCR3), NKp44 (NCR2), DNAM1(CD226), CRTAM, CD27, NTB-A (SLAMF6), PSGL1, CD96 (Tactile), CD100(SEMA4D), NKp80 (KLRF1, CLECSC), SLAMF7 (CRACC, CS1, CD319), and CD244(2B4, SLAMF4).

Combination Therapies Comprising Immunotherapeutic Agents, and ExemplaryImmunotherapeutic Agents

In some embodiments a method of treating cancer provided hereincomprises administering to the individual an effective amount of animmunotherapeutic agent (e.g., at least one immunotherapeutic agent,such as at least two, at least three, or at least four immunotherapeuticagents), i.e., in combination with an agent that blocks the interactionbetween CD47 (e.g., hCD47) and SIRPα (e.g., a polypeptide describedherein) and a chemotherapeutic agent described herein (e.g., at leastone chemotherapeutic agent, such as at least two, at least three, or atleast four chemotherapeutic agents).

In some embodiments, an immunotherapeutic agent refers to anytherapeutic that targets the immune system and promotes a therapeuticredirection of the immune system, such as a modulator of a costimulatorypathway, cancer vaccine, recombinantly modified immune cell, etc.Exemplary and non-limiting immunotherapeutic agents are described infra.In some embodiments, the immunotherapeutic agent is or comprises anantibody. Exemplary targets of immunotherapeutic antibodies are known inthe art and include, without limitation, BDCA2, BDCA4, ILT7, LILRB1,LILRB2, LILRB3, LILRB4, LILRB5, Siglec-3, Siglec-7, Siglec-9, Siglec-10,Siglec-15, FGL-1, CD200, CD200R, CSF-1R, CD24, CD40, CD40L, CD163,CD206, DEC205, CD47, CD123, arginase, IDO, TDO, AhR, EP2, COX-2, CCR2,CCR-7, CXCR1, CX3CR1, CXCR2, CXCR3, CXCR4, CXCR7, TGF-β RI, TGF-β RH,c-Kit, CD244, L-selectin/CD62L, CD11b, CD11 c, CD68, 41BB, CTLA4, PD1,PD-L1, PD-L2, TIM-3, BTLA, VISTA, LAG-3, CD28, OX40, GITR, CD137, CD27,HVEM, CCR4, CD25, CD103, Klrgl, Nrpl, CD278, Gpr83, TIGIT, CD154, CD160,TNFR2, PVRIG, DNAM, and ICOS.

Immunotherapeutic agents that are approved or in late-stage clinicaltesting include, without limitation, ipilimumab, pembrolizumab,nivolumab, atezolizumab, avelumab, durvalumab, and the like. In certainembodiments, the agent that blocks the interaction between CD47 andSIRPα (such as a polypeptide described herein) is administered incombination with an inhibitor of the PD-L1/PD-1 pathway, e.g., anantibody, a small molecule, or polypeptide that blocks the interactionbetween PD-L1 and PD-1 (e.g., by binding to PD-1 or PD-L1). In someembodiments, the inhibitor of the PD-L1/PD-1 pathway is an antisensepolynucleotide. In some embodiments, the inhibitor of the PD-L1/PD-1pathway is an anti-PD-L1 or anti-PD-1 antagonist antibody (e.g., ananti-PD-1 or anti-PD-L1 antagonist antibody described elsewhere herein).As demonstrated herein, combined administration of an agent that blocksthe interaction between CD47 and SIRPα (such as a polypeptide describedherein) and an inhibitor of the PD-L1/PD-1 pathway can result insynergistic anti-tumor activity. In some embodiments, theimmunotherapeutic agent is or comprises a vaccine, oncolytic virus,adoptive cell therapy, cytokine, or small molecule immunotherapeuticagent. Examples of such immunotherapeutic agents are known in the art.For example, adoptive cell therapies and therapeutics can includewithout limitation chimeric antigen receptor T-cell therapy (CAR-T),tumor infiltrating lymphocytes (TILs), TCR engineered T cells, TCRengineered NK cell, and macrophage cell products. Vaccines can includewithout limitation polynucleotide vaccines, polypeptide vaccines, orcell-based (e.g., tumor or dendritic cell-based) vaccines. Variouscytokines useful for the treatment of cancer are known and includewithout limitation IL-2, IL-15, IL-7, IL-10, IL-12, IL21, TNFa, IFNs,GM-CSF, and engineered cytokine mutants. Small moleculeimmunotherapeutic agents can include without limitation IDO/TDOinhibitors, AhR inhibitors, arginase inhibitors, A2a R inhibitors, TLRagonists, STING agonists, and Rig-1 agonists.

In some embodiments where the agent that blocks the interaction betweenCD47 and SIRPα (such as a polypeptide described herein) and thechemotherapeutic agent (e.g., at least one chemotherapeutic agent) areadministered in combination with further agent(s) described herein(e.g., therapeutic antibodies, small molecule inhibitors,immunotherapeutic agents, etc.), the further agent(s) are from differentclasses and/or exert their anti-cancer effects via different mechanismsof action. For example, in some embodiments, the method of treatingcancer comprises administering an agent that blocks the interactionbetween CD47 and SIRPα (such as a polypeptide described herein) incombination with a chemotherapeutic agent (including, but not limited tothose described herein) and a therapeutic antibody (including, but notlimited to those described herein, e.g., an anti-HER2 antibody). In someembodiments, the agent that blocks the interaction between CD47 andSIRPα (such as a polypeptide described herein) is administered incombination with a chemotherapeutic agent (including, but not limited tothose described herein) and a small molecule inhibitor (including, butnot limited to those described herein). Other combinations are alsocontemplated.

In some embodiments, the agent that blocks the interaction between CD47and SIRPα (such as a polypeptide described herein) is administered incombination with one or more agents including, without limitation, e.g.,anti-diarrheal agents, anti-emetic agents, analgesics, opioids and/ornon-steroidal anti-inflammatory agent.

Combination Therapies that Comprise Additional Mode(s) of Therapy

In some embodiments, the agent that blocks the interaction between CD47and SIRPα (such as a polypeptide described herein) is administered incombination with at least one chemotherapy agent and one or moreadditional modes of therapy. In some embodiments, the one or moreadditional modes therapy comprises radiotherapy (e.g., gamma-rays,X-rays, and/or the directed delivery of radioisotopes to tumor cells,microwaves, UV radiation, or gene therapy. For example, therapeuticgenes for gene therapy include, but are not limited to, an antisenseversion of an inducer of cellular proliferation (oncogene), an inhibitorof cellular proliferation (tumor suppressor), or an inducer ofprogrammed cell death (pro-apoptotic gene). In some embodiments, any oneor more of the combination therapies described herein are administeredin conjunction with a surgery (e.g., resection).

Exemplary Therapeutic Combinations

In some embodiments, the method of treating cancer comprisesadministering an agent that blocks the interaction between CD47 (e.g.,hCD47) and SIRPα (e.g., hSIRPα) in combination with nivolumab and one oragents selected from: lenalidomide, ibrutinib, palbociclib,enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib,erlotinib, bortezomib, enzalutamide, cyclophosphamide, carboplatin,cisplatin, oxaliplatin, 5-fluorouracil, 6-mercaptopurine, cytarabine,gemcitabine, methotrexate, bleomycin, daunorubicin, doxorubicin,docetaxel, estramustine, paclitaxel, vinblastine, etoposide, irinotecan,teniposide, topotecan, prednisone, methylprednisolone, anddexamethasone. In some embodiments, the agent that blocks theinteraction between CD47 and SIRPα is a polypeptide described herein(e.g., a fusion polypeptide comprising a SIRPα d1 domain variant and anFc variant).

In some embodiments, the method of treating cancer comprisesadministering an agent that blocks the interaction between CD47 (e.g.,hCD47) and SIRPα (e.g., hSIRPα) in combination with pembrolizumab andone or agents selected from: lenalidomide, ibrutinib, palbociclib,enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib,erlotinib, bortezomib, enzalutamide, cyclophosphamide, carboplatin,cisplatin, oxaliplatin, 5-fluorouracil, 6-mercaptopurine, cytarabine,gemcitabine, methotrexate, bleomycin, daunorubicin, doxorubicin,docetaxel, estramustine, paclitaxel, vinblastine, etoposide, irinotecan,teniposide, topotecan, prednisone, methylprednisolone, anddexamethasone. In some embodiments, the agent that blocks theinteraction between CD47 and SIRPα is a polypeptide described herein(e.g., a fusion polypeptide comprising a SIRPα d1 domain variant and anFc variant).

In some embodiments, the method of treating cancer comprisesadministering an agent that blocks the interaction between CD47 (e.g.,hCD47) and SIRPα (e.g., hSIRPα) in combination with trastuzumab and oneor agents selected from: lenalidomide, ibrutinib, palbociclib,enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib,erlotinib, bortezomib, enzalutamide, cyclophosphamide, carboplatin,cisplatin, oxaliplatin, 5-fluorouracil, 6-mercaptopurine, cytarabine,gemcitabine, methotrexate, bleomycin, daunorubicin, doxorubicin,docetaxel, estramustine, paclitaxel, vinblastine, etoposide, irinotecan,teniposide, topotecan, prednisone, methylprednisolone, anddexamethasone. In some embodiments, the agent that blocks theinteraction between CD47 and SIRPα is a polypeptide described herein(e.g., a fusion polypeptide comprising a SIRPα d1 domain variant and anFc variant).

In some embodiments, the method of treating cancer comprisesadministering an agent that blocks the interaction between CD47 (e.g.,hCD47) and SIRPα (e.g., hSIRPα) in combination with bevacizumab and oneor agents selected from: lenalidomide, ibrutinib, palbociclib,enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib,erlotinib, bortezomib, enzalutamide, cyclophosphamide, carboplatin,cisplatin, oxaliplatin, 5-fluorouracil, 6-mercaptopurine, cytarabine,gemcitabine, methotrexate, bleomycin, daunorubicin, doxorubicin,docetaxel, estramustine, paclitaxel, vinblastine, etoposide, irinotecan,teniposide, topotecan, prednisone, methylprednisolone, anddexamethasone. In some embodiments, the agent that blocks theinteraction between CD47 and SIRPα is a polypeptide described herein(e.g., a fusion polypeptide comprising a SIRPα d1 domain variant and anFc variant).

In some embodiments, the method of treating cancer comprisesadministering an agent that blocks the interaction between CD47 (e.g.,hCD47) and SIRPα (e.g., hSIRPα) in combination with rituximab and one oragents selected from: lenalidomide, ibrutinib, palbociclib,enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib,erlotinib, bortezomib, enzalutamide, cyclophosphamide, carboplatin,cisplatin, oxaliplatin, 5-fluorouracil, 6-mercaptopurine, cytarabine,gemcitabine, methotrexate, bleomycin, daunorubicin, doxorubicin,docetaxel, estramustine, paclitaxel, vinblastine, etoposide, irinotecan,teniposide, topotecan, prednisone, methylprednisolone, anddexamethasone. In some embodiments, the agent that blocks theinteraction between CD47 and SIRPα is a polypeptide described herein(e.g., a fusion polypeptide comprising a SIRPα d1 domain variant and anFc variant).

In some embodiments, the method of treating cancer comprisesadministering an agent that blocks the interaction between CD47 (e.g.,hCD47) and SIRPα (e.g., hSIRPα) in combination with pertuzumab and oneor agents selected from: lenalidomide, ibrutinib, palbociclib,enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib,erlotinib, bortezomib, enzalutamide, cyclophosphamide, carboplatin,cisplatin, oxaliplatin, 5-fluorouracil, 6-mercaptopurine, cytarabine,gemcitabine, methotrexate, bleomycin, daunorubicin, doxorubicin,docetaxel, estramustine, paclitaxel, vinblastine, etoposide, irinotecan,teniposide, topotecan, prednisone, methylprednisolone, anddexamethasone. In some embodiments, the agent that blocks theinteraction between CD47 and SIRPα is a polypeptide described herein(e.g., a fusion polypeptide comprising a SIRPα d1 domain variant and anFc variant).

In some embodiments, the method of treating cancer comprisesadministering an agent that blocks the interaction between CD47 (e.g.,hCD47) and SIRPα (e.g., hSIRPα) in combination with denosumab and one oragents selected from: lenalidomide, ibrutinib, palbociclib,enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib,erlotinib, bortezomib, enzalutamide, cyclophosphamide, carboplatin,cisplatin, oxaliplatin, 5-fluorouracil, 6-mercaptopurine, cytarabine,gemcitabine, methotrexate, bleomycin, daunorubicin, doxorubicin,docetaxel, estramustine, paclitaxel, vinblastine, etoposide, irinotecan,teniposide, topotecan, prednisone, methylprednisolone, anddexamethasone. In some embodiments, the agent that blocks theinteraction between CD47 and SIRPα is a polypeptide described herein(e.g., a fusion polypeptide comprising a SIRPα d1 domain variant and anFc variant).

Exemplary Cancers

In some embodiments, the cancer treated by a method provided herein isbreast cancer, lung cancer, adenocarcinoma of the lung, squamous celllung cancer, small cell lung cancer (SCLC), non-small cell lung cancer(NSCLC), head and neck cancer, mesothelioma, brain cancer, brain tumor,abdominal cancer, colon cancer, colorectal cancer, esophageal cancer,parapharyngeal cancer, gastrointestinal cancer, glioma, liver cancer,gastric cancer, oral cancer, tongue cancer, neuroblastoma, osteosarcoma,ovarian cancer, renal cancer, urinary bladder cancer, urinary tractcancer, pancreatic cancer, retinoblastoma, cervical cancer, uterinecancer, Wilm's tumor, multiple myeloma, skin cancer, lymphoma, leukemia,blood cancer, thyroid cancer, bone cancer, adenocystic tumor,chondrosar-coma, pancreatic islet cell tumor, neuroendocrine tumor,prostate cancer, glioblastoma, endometrial carcinoma, endometrialcancer, leiomyosarcoma, gall bladder cancer, hepatocellular cancer, amelanoma, or a solid tumor.

In some embodiments, the cancer treated by a method provided herein is ahematological cancer. In some embodiments, the hematological cancer ismultiple myeloma, or a leukemia, including, but not limited to, e.g.,acute or chronic myelogenous leukemia acute or chronic lymphoblasticleukemia, acute lymphocytic leukemia (ALL) chronic lymphocytic leukemia(CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML),myelodysplastic syndrome (MDS), chronic myeloid leukemia (CIVIL), hairycell leukemia, chronic myelomonocytic leukemia (CMML), Juvenilemyelomonocytic leukemia (JMML), large granular lymphocytic (LGL)leukemia, plasmacytoma, blastic plasmacytoid dendritic cell neoplasm(BPDCN), B-cell prolymphocytic leukemia (B-PLL), T-cell prolymphocyticleukemia (T-PLL), multiple myeloma (MM), and Non-Hodgkin lymphomas (suchas diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, mantle celllymphoma (MCL), peripheral T-cell lymphoma (PTCL), lymphoplasmacyticlymphoma, Waldenström macroglobulinemia, marginal zone lymphoma (MZL)and follicular lymphoma (FL).

Methods of Treating Leukemia

In some embodiments, provided is a method of treating leukemia (e.g.,acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL),small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML),myelodysplastic syndrome (MDS), chronic myeloid leukemia (CIVIL), hairycell leukemia, chronic myelomonocytic leukemia (CMML), Juvenilemyelomonocytic leukemia (JMML), large granular lymphocytic (LGL)leukemia, blastic plasmacytoid dendritic cell neoplasm (BPDCN), B-cellprolymphocytic leukemia (B-PLL), T-cell prolymphocytic leukemia (T-PLL),multiple myeloma (MM), and Non-Hodgkin lymphomas (such as diffuse largeB-cell lymphoma (DLBCL), Burkitt lymphoma, mantle cell lymphoma (MCL),peripheral T-cell lymphoma (PTCL), lymphoplasmacytic lymphoma,Waldenström macroglobulinemia, marginal zone lymphoma (MZL) andfollicular lymphoma (FL)), in an individual (e.g., a human individual)that comprises administering to the individual an effective amount of(a) an agent that blocks the interaction between CD47 (e.g., hCD47) andSIRPα (e.g., hSIRPα) and (b) a Bcl2 inhibitor. In some embodiments, theBcl2 inhibitor is venetoclax (also known as ABT-199), ABT-737,navitoclax (also known as ABT-263), BCL201, or AZD-0466. In someembodiments, the agent is a polypeptide (e.g., fusion polypeptide)comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variantdescribed herein) and an Fc domain variant (e.g., an Fc domain variantdescribed herein). In some embodiments, the polypeptide (e.g., fusionpolypeptide) comprises a SIRPα D1 domain variant that comprises theamino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In someembodiments, the Fc domain variant is (i) a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat. Insome embodiments, the polypeptide (e.g., fusion polypeptide)administered to the individual comprises the amino acid sequence of SEQID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide(e.g., fusion polypeptide) forms a homodimer. In some embodiments, thepolypeptide (e.g., fusion polypeptide) and the Bcl2 inhibitor (e.g.,venetoclax) are administered simultaneously, concurrently, orsequentially.

Bcl2 inhibitors are a class of anticancer drugs that are believed toexert their cytotoxic effects by competing with proapoptotic Bcl2s tooccupy BH3 docking grooves on the surfaces of antiapoptotic familymembers. By binding to one or more Bcl2 family members, these inhibitorsinduce apoptosis by mimicking the activity of natural antagonists ofBCL-2 and other related proteins and restore apoptosis in tumor cells.

Venetoclax (also known as GDC-0199, ABT-199, and RG7601) is an exemplaryselective Bcl2 inhibitor used in the methods described herein.Venetoclax is a light yellow to dark yellow solid with the empiricalformula C₄₅H₅₀ClN₇O₇S and a molecular weight of 868.44 g/mol. Venetoclaxhas very low aqueous solubility. Venetoclax is described chemically as4-(4-{[2-(4-chlorophenyl)-4,4dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide)and has the following chemical structure:

The CAS Registry Number for venetoclax is 1257044-40-8. Venetoclax isadministered orally and is sold under the trade names Venclexta andVenclyxto. Complete information about venetoclax preparation,dispensing, dosage, and administration schedule can be found in thelocal package insert (for the United States, see, e.g.,www(dot)accessdata(dot)fda(dot)gov/drugsatfda_docs/label/2016/208573s000lbl(dot)pdf;for Europe, see, e.g.,www(dot)ema(dot)europa(dot)eu/en/medicines/human/EPAR/venclyxto#product-information-section).In some embodiments, the venetoclax is administered in accordance withthe dosing and frequency recommended in the local package insert.

ABT-737 is another exemplary selective Bcl2 inhibitor used in themethods described herein. ABT-737, which inhibits both Bcl2 and Bcl-xL,has the empirical formula C₄₂H₄₅ClN₆O₅S₂ and a molecular weight of813.43 g/mol. The CAS Registry Number for ABT-737 is 852-808-04-9.ABT-737 is described chemically as4-{4-[(4′-Chloro-2-biphenylyl)methyl]-1-piperazinyl}-N-[(4-{[(2R)-4-(dimethylamino)-1-(phenylsulfanyl)-2-butanyl]amino}-3-nitrophenyl)sulfonyl]benzamide and has the following chemical structure:

Another exemplary selective Bcl2 inhibitor used in the methods describedherein is navitoclax (also known as ABT-263). Navitoclax, which inhibitsboth Bcl2, Bcl-xL, and Bcl-w, has the empirical formula C₄₇H₅₅ClF₃N₅O₆S₃and a molecular weight of 974.6 g/mol. The CAS Registry Number fornavitoclax is 923564-51-6. ABT-737 is described chemically as4-[4-[[2-(4-chlorophenyl)-5,5-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-morpholin-4-yl-1-phenylsulfanylbutan-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonylbenzamide and has the chemical structure provided below.Additional details regarding navitoclax are provided in, e.g., Tse etal. (2008) Cancer Res. 68(9): 3421-3429.

Another exemplary selective Bcl2 inhibitor used in the methods describedherein is S55746 (also known as BCL201 and Servier-1). S55746 occupiesthe hydrophobic groove of BCL-2. Its selectivity profile demonstrates nosignificant binding to MCL-1, BFL-1 S55746 occupies the hydrophobicgroove of BCL-2. Its selectivity profile demonstrates no significantbinding to MCL-1, BFL-1 (BCL2A1/A1) and poor affinity for BCL-XL. S55746has no cytotoxic activity on BCL-XL-dependent cells, such as platelets(see, e.g., Casara et al. (2008) Oncotarget. 9(28): 29975-20088). S55746has the empirical formula C₄₃H₄₂N₄O₆ and a molecular weight of 710.82g/mol. The CAS Registry Number for S55746 is 1448584-12-0. S55746 isdescribed chemically as(S)-N-(4-hydroxyphenyl)-3-(6-(3-(morpholinomethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)benzo[d][1,3]dioxol-5-yl)-N-phenyl-5,6,7,8-tetrahydroindolizine-1-carboxamideand has the following chemical structure:

Methods of Treating Solid Tumor

In some embodiments, provided is a method of treating solid tumor in anindividual (e.g., a human individual) that comprises administering tothe individual an effective amount of (a) an agent that blocks theinteraction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) and (b)a platinum-based chemotherapy agent. In some embodiments, the solidtumor is colon cancer (e.g., colon carcinoma), lung cancer, head andneck cancer, esophageal cancer, breast cancer, bladder cancer, ovariancancer, cervical cancer, testicular cancer, brain tumor, mesothelioma,or neuroblastoma. In some embodiments, the platinum-based chemotherapyagent is carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatintetranitrate, phenanthriplatin, picoplatin, and/or satraplatin. In someembodiments, the platinum-based chemotherapy agent is cisplatin. In someembodiments, the agent is a polypeptide (e.g., fusion polypeptide)comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variantdescribed herein) and an Fc domain variant (e.g., an Fc domain variantdescribed herein). In some embodiments, the polypeptide (e.g., fusionpolypeptide) comprises a SIRPα D1 domain variant that comprises theamino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In someembodiments, the Fc domain variant is (i) a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat. Insome embodiments, the polypeptide (e.g., fusion polypeptide)administered to the individual comprises the amino acid sequence of SEQID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide(e.g., fusion polypeptide) forms a homodimer. In some embodiments, thepolypeptide (e.g., fusion polypeptide) and the platinum-basedchemotherapy agent (e.g., cisplatin) are administered simultaneously,concurrently, or sequentially.

Platinum agents (such as carboplatin, cisplatin, oxaliplatin,nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, andsatraplatin) are widely used antitumor drugs that cause crosslinking ofDNA as monoadduct, interstrand crosslinks, intrastrand crosslinks or DNAprotein crosslinks. Platinum agents typically act on the adjacent N-7position of guanine, forming a 1, 2 intrastrand crosslink (Poklar et al.(1996). Proc. Natl. Acad. Sci. U.S.A. 93 (15): 7606-11; Rudd et al.(1995). Cancer Chemother. Pharmacol. 35 (4): 323-6). The resultantcrosslinking inhibits DNA repair and/or DNA synthesis in cancer cells.

Cisplatin is an exemplary platinum coordination compound used in themethods described herein. The chemical name for cisplatin isdichloroplatinum diammoniate, and cisplatin has the following structuralformula:

Cisplatin is an inorganic and water-soluble platinum complex with themolecular formula of Pt(NH₃)₂Cl₂ and a molecular weight of 300.046.After undergoing hydrolysis, it reacts with DNA to produce both intraand interstrand crosslinks. These crosslinks appear to impairreplication and transcription of DNA. The cytotoxicity of cisplatincorrelates with cellular arrest in the G2 phase of the cell cycle.Cisplatin, which has been assigned the CAS Registry No. 15663-27-1, iscommercially available as PLATINOL®, PLATINOL®-AQ, CDDP, CISPLAN,CISPLAT, PLATIKEM, PLATIONCO, PRACTICIS, PLATICIS, BLASTOLEM, CISMAX,CISPLAN, CISPLATINUM, CISTEEN, DUPLAT, KEMOPLAT, ONCOPLATIN-AQ,PLATINEX, PLATIN, TEVAPLATIN, and others. Complete information aboutcisplatin preparation, dispensing, dosage, and administration schedulecan be found in local package insert (for the United States, see, e.g.,www(dot)accessdata(dot)fda(dot)gov/drugsatfda_docs/label/2011/018057s080lbl(dot)pdfandwww(dot)accessdata(dot)fda(dot)gov/drugsatfda_docs/label/2015/018057s083lbl(dot)pdf).In some embodiments, the cisplatin is administered in accordance withthe dosing and frequency recommended in the local package insert.

Carboplatin is another exemplary platinum coordination compound used inthe methods described herein. The chemical name for carboplatin isplatinum, diammine [1,1cyclobutane-dicarboxylato(2-)-0,0]-,(SP-4-2), andcarboplatin has the following structural formula:

Carboplatin is a water-soluble platinum complex with the molecularformula of C₆H₁₂N₂O₄Pt and a molecular weight of 373.26. Carboplatin hasbeen assigned the CAS Registration Number 41575-94-4, and its mechanismof action is similar to that of cisplatin. Carboplatin is typicallyprescribed more commonly than cisplatin. Carboplatin is commerciallyavailable as PARAPLATIN®, BLASTOCARB®, BLASTOPLATIN®, CARBOKEM®,CARBOMAX®, PARAPLATIN®, CARBOPA®, KARPLAT®, and others. Completeinformation about carboplatin preparation, dispensing, dosage, andadministration schedule can be found in local package insert (for theUnited States, see, e.g.,www(dot)accessdata(dot)fda(dot)gov/drugsatfda_docs/label/2010/020452s005lbl(dot)pdfandwww(dot)accessdata.fda(dot)gov/drugsatfda_docs/label/2012/077139Orig1s016lbl(dot)pdf).In some embodiments, the carboplatin is administered in accordance withthe dosing and frequency recommended in the local package insert.

In some embodiments, provided is a method of treating solid tumor in anindividual (e.g., a human individual) that comprises administering tothe individual an effective amount of (a) an agent that blocks theinteraction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) ananti-HER 2 antibody, and (c) an anti-PDL1 antibody. In some embodimentsthe anti-HER2 antibody is trastuzumab (CAS Registry No. 180288-69-1). Insome embodiments the anti-PDL1 antibody is atezolizumab (CAS RegistryNo. 1380723-44-3), avelumab (CAS Registry No. 1537032-82-8), ordurvalumab (CAS Registry No. 1428935-60-7). In some embodiments, theagent is a polypeptide (e.g., fusion polypeptide) comprising a SIRPα D1domain variant (e.g., a SIRPα D1 domain variant described herein) and anFc domain variant (e.g., an Fc domain variant described herein). In someembodiments, the polypeptide (e.g., fusion polypeptide) comprises aSIRPα D1 domain variant that comprises the amino acid sequence of SEQ IDNO: 81 or SEQ ID NO: 85. In some embodiments, the Fc domain variant is(i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297Amutations, wherein numbering is according to the EU index of Kabat; (ii)a human IgG2 Fc region comprising A330S, P331S, and N297A mutations,wherein numbering is according to the EU index of Kabat; (iii) a humanIgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236mutations, wherein numbering is according to the EU index of Kabat; or(iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A,delG236, and N297A mutations, wherein numbering is according to the EUindex of Kabat. In some embodiments, the polypeptide (e.g., fusionpolypeptide) administered to the individual comprises the amino acidsequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, thepolypeptide (e.g., fusion polypeptide) forms a homodimer. In someembodiments, the polypeptide (e.g., fusion polypeptide), the anti-HER2antibody, the anti-PD-L1 antibody (e.g., an anti PD-L1 antagonistantibody) are administered simultaneously, concurrently, orsequentially. In some embodiments, the solid tumor is colon cancer, lungcancer, head and neck cancer, esophageal cancer, breast cancer, bladdercancer, ovarian cancer, cervical cancer, testicular cancer, endometrialcancer, liver cancer, gastric cancer, gastroesophageal junction cancer,brain tumor, mesothelioma, or neuroblastoma. In some embodiments, thesolid tumor is HER2⁺ solid tumor. In some embodiments, the solid tumoris colon cancer (e.g., HER2⁺ colon cancer).

Methods of Treating Gastric or Gastroesophageal Junction (GEJ) Cancer

In some embodiments, provided is a method of treating gastric cancer orgastroesophageal junction (GEJ) cancer in an individual (e.g., a humanindividual) that comprises administering to the individual an effectiveamount of (a) an agent that blocks the interaction between CD47 (e.g.,hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-HER 2 antibody, (c) ananti-VEGFR2 antibody, and (d) paclitaxel. In some embodiments theanti-HER2 antibody is trastuzumab (CAS Registry No. 180288-69-1). Insome embodiments the anti-VEGFR2 antibody is ramucirumab (CAS RegistryNo. 947687-13-0). In some embodiments, the agent is a polypeptide (e.g.,fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPαD1 domain variant described herein) and an Fc domain variant (e.g., anFc domain variant described herein). In some embodiments, thepolypeptide (e.g., fusion polypeptide) comprises a SIRPα D1 domainvariant that comprises the amino acid sequence of SEQ ID NO: 81 or SEQID NO: 85. In some embodiments, the Fc domain variant is (i) a humanIgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations,wherein numbering is according to the EU index of Kabat; (ii) a humanIgG2 Fc region comprising A330S, P331S, and N297A mutations, whereinnumbering is according to the EU index of Kabat; (iii) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, and delG236 mutations,wherein numbering is according to the EU index of Kabat; or (iv) a humanIgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat. Insome embodiments, the polypeptide (e.g., fusion polypeptide)administered to the individual comprises the amino acid sequence of SEQID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide(e.g., fusion polypeptide) forms a homodimer. In some embodiments, thepolypeptide (e.g., fusion polypeptide), the anti-HER2 antibody, theanti-VEGFR2 antibody, and the paclitaxel are administeredsimultaneously, concurrently, or sequentially. In some embodiments, thepolypeptide (e.g. fusion polypeptide) is administered to the individualat a dose of 10 mg/kg once a week or 15 mg/kg once a week. In someembodiments, the individual receiving treatment has gastric or GEJadenocarcinoma. In some embodiments, the individual receiving treatmenthas HER2⁺ gastric cancer or HER2⁺ GEJ cancer (e.g., a HER2-overexpessinggastric or GEJ cancer). In some embodiments, the HER2⁺ gastric cancer orHER2⁺ GEJ cancer is advanced and/or metastatic. In some embodiments, theindividual receiving treatment has gastric or GEJ cancer that hasprogressed during or after prior treatment(s) comprising anti-HER2antibody (e.g., trastuzumab). In some embodiments, the individualreceiving treatment has gastric or GEJ cancer that has progressed duringor after prior treatment(s) comprising anti-HER2 antibody (e.g.,trastuzumab) and a fluoropyrimidine. In some embodiments, the individualreceiving treatment has gastric or GEJ cancer that has progressed duringor after prior treatments(s) comprising anti-HER2 antibody (e.g.,trastuzumab) and a platinum-based chemotherapeutic agent. In someembodiments, the individual receiving treatment has gastric or GEJcancer (e.g., HER2⁺ gastric cancer or GEJ cancer) that has progressedduring or after prior treatment(s) comprising anti-HER2 antibody (e.g.,trastuzumab) and/or a fluoropyrimidine, and/or a platinum-basedchemotherapeutic agent. In some embodiments, the individual failed(e.g., relapsed after or did not respond to) prior therapy with ananti-HER2 antibody, with an anti-HER2 antibody and a fluoropyrimidine,or with an anti-HER2 antibody and a platinum-based chemotherapy agent.In some embodiments, the fluoropyrimidine was fluorouracil (also knownas 5-fluorouracil). In some embodiments, treatment with the polypeptide,the anti-HER2 antibody, the anti-VEGFR2 antibody, and the paclitaxeldoes not result in adverse effects. In some embodiments, treatment withthe polypeptide, the anti-HER2 antibody, the anti-VEGFR2 antibody, andthe paclitaxel results in only low grade adverse effects.

In some embodiments, provided is a method of treating gastric cancer orgastroesophageal junction (GEJ) cancer in an individual (e.g., a humanindividual) that comprises administering to the individual an effectiveamount of (a) an agent that blocks the interaction between CD47 (e.g.,hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-PD-1 antibody (e.g., ananti-PD-1 antagonist antibody), (c) an anti-HER2 antibody, (d)5-fluorouracil and (e) a platinum-based chemotherapeutic agent. In someembodiments, provided is a method of treating gastric cancer orgastroesophageal junction (GEJ) cancer in an individual (e.g., a humanindividual) that comprises administering to the individual an effectiveamount of (a) an agent that blocks the interaction between CD47 (e.g.,hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-PD-1 antibody (e.g., ananti-PD-1 antagonist antibody), (c) an anti-HER2 antibody, (d)capecitabine, and (e) a platinum-based chemotherapeutic agent. In someembodiments the anti-PD-1 antibody is pembrolizumab (CAS Registry No.1374853-91-4). In some embodiments the anti-HER2 antibody is trastuzumab(CAS Registry No. 180288-69-1). In some embodiments, the platinum-basedchemotherapeutic agent is cisplatin. In some embodiments, the agent is apolypeptide (e.g., fusion polypeptide) comprising a SIRPα D1 domainvariant (e.g., a SIRPα D1 domain variant described herein) and an Fcdomain variant (e.g., an Fc domain variant described herein). In someembodiments, the polypeptide (e.g., fusion polypeptide) comprises aSIRPα D1 domain variant that comprises the amino acid sequence of SEQ IDNO: 81 or SEQ ID NO: 85. In some embodiments, the Fc domain variant is(i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297Amutations, wherein numbering is according to the EU index of Kabat; (ii)a human IgG2 Fc region comprising A330S, P331S, and N297A mutations,wherein numbering is according to the EU index of Kabat; (iii) a humanIgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236mutations, wherein numbering is according to the EU index of Kabat; or(iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A,delG236, and N297A mutations, wherein numbering is according to the EUindex of Kabat. In some embodiments, the polypeptide (e.g., fusionpolypeptide) administered to the individual comprises the amino acidsequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, thepolypeptide (e.g., fusion polypeptide) forms a homodimer. In someembodiments, the polypeptide (e.g., fusion polypeptide), the anti-PD-1antibody, the anti-HER2 antibody, the 5-fluorouracil, and theplatinum-based chemotherapeutic agent are administered simultaneously,concurrently, or sequentially. In some embodiments, the polypeptide(e.g., fusion polypeptide), the anti-PD-1 antibody, the anti-HER2antibody, the capecitabine, and the platinum-based chemotherapeuticagent are administered simultaneously, concurrently, or sequentially. Insome embodiments, the individual receiving treatment hasHER2-overexpressing gastric cancer or HER2-overexpressing GEJ cancer. Insome embodiments, the gastric cancer or the GEJ cancer is advancedand/or metastatic. In some embodiments, the individual has not receivedprior treatment for gastric cancer or the GEJ cancer.

Methods of Treating Head and Neck Cancer

In some embodiments, provided is a method of treating head and neckcancer (e.g., head and neck cancer squamous cell carcinoma or HNSCC) inan individual (e.g., a human individual) that comprises administering tothe individual an effective amount of (a) an agent that blocks theinteraction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) aPD-1 inhibitor, (c) an antimetabolite, and (d) a platinum-based agent.In some embodiments, the PD-1 inhibitor is a small molecule inhibitor,an antisense nucleotide, or a peptide. In some embodiments, the PD-1inhibitor is an anti-PD-1 antibody. In some embodiments, the anti-PD-1antibody is pembrolizumab, nivolumab, pidilizumab, cemiplimab, orBMS-936559. In some embodiments, the anti-PD-1 antibody is pembrolizumab(CAS Registry No. 1374853-91-4). In some embodiments, the antimetaboliteis 5-fluorouracil, 6-mercaptopurine, capecitabine, cytarabine,floxuridine, fludarabine, gemcitabine, hydroxycarbamide, methotrexate,pemetrexed, phototrexate. In some embodiments, the antimetabolite is5-fluorouracil. In some embodiments, the platinum-based chemotherapyagent is carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatintetranitrate, phenanthriplatin, picoplatin, or satraplatin. In someembodiments, the platinum-based chemotherapy agent is cisplatin orcarboplatin. In some embodiments, the agent is a polypeptide (e.g.,fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPαD1 domain variant described herein) and an Fc domain variant (e.g., anFc domain variant described herein). In some embodiments, thepolypeptide (e.g., fusion polypeptide) comprises a SIRPα D1 domainvariant that comprises the amino acid sequence of SEQ ID NO: 81 or SEQID NO: 85. In some embodiments, the Fc domain variant is (i) a humanIgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations,wherein numbering is according to the EU index of Kabat; (ii) a humanIgG2 Fc region comprising A330S, P331S, and N297A mutations, whereinnumbering is according to the EU index of Kabat; (iii) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, and delG236 mutations,wherein numbering is according to the EU index of Kabat; or (iv) a humanIgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat. Insome embodiments, the polypeptide (e.g., fusion polypeptide)administered to the individual comprises the amino acid sequence of SEQID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide(e.g., fusion polypeptide) forms a homodimer. In some embodiments, thepolypeptide (e.g., fusion polypeptide), the PD-1 inhibitor (e.g., ananti-PD-1 antibody, e.g., pembrolizumab), the antimetabolite (e.g.,5-fluorouracil), and the platinum-based chemotherapeutic agent (e.g.,cisplatin or carboplatin) are administered simultaneously, concurrently,or sequentially. In some embodiments, the polypeptide (e.g. fusionpolypeptide) is administered to the individual at a dose of 10 mg/kgonce a week or 15 mg/kg once a week. In some embodiments, the individualreceiving treatment has HNSCC. In some embodiments, the HNSCC isadvanced and/or metastatic HNSCC. In some embodiments, the HNSCC isunresectable and/or recurrent. In some embodiments, the individual hasnot received prior treatment for head and neck cancer (e.g., HNSCC). Insome embodiments, treatment with the polypeptide, the PD-1 inhibitor(e.g., pembrolizumab), the antimetabolite (e.g., 5-fluorouracil), andthe platinum-based chemotherapeutic agent (e.g., cisplatin orcarboplatin) does not result in adverse effects. In some embodimentstreatment with the polypeptide, the PD-1 inhibitor (e.g.,pembrolizumab), the antimetabolite (e.g., 5-fluorouracil), and theplatinum-based chemotherapeutic agent (e.g., cisplatin or carboplatin)results in only low grade adverse effects.

Combination Cancer Therapies Comprising an Anti-TROP2 Antibody

In some embodiments, provided is a method of treating cancer in anindividual (e.g., a human individual) that comprises administering tothe individual an effective amount of (a) an agent that blocks theinteraction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) and (b)an anti-TROP2 antibody. In some embodiments, the anti-TROP2 antibody isRS7, which is described in U.S. Pat. No. 10,179,171, the contents ofwhich are incorporated herein in their entirety. In some embodiments,the anti-TROP2 antibody is conjugated to a drug (i.e., an antibody-drugconjugate or “ADC”). In some embodiments, the anti-TROP2 ADC isSacituzumab govitecan (also known as hRS7-SN38 or IMMU-132), which isdescribed in US 2017/0281791, the contents of which are incorporatedherein by reference in their entirety. In some embodiments, the agent isa polypeptide (e.g., fusion polypeptide) comprising a SIRPα D1 domainvariant (e.g., a SIRPα D1 domain variant described herein) and an Fcdomain variant (e.g., an Fc domain variant described herein). In someembodiments, the polypeptide (e.g., fusion polypeptide) comprises aSIRPα D1 domain variant that comprises the amino acid sequence of SEQ IDNO: 81 or SEQ ID NO: 85. In some embodiments, the Fc domain variant is(i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297Amutations, wherein numbering is according to the EU index of Kabat; (ii)a human IgG2 Fc region comprising A330S, P331S, and N297A mutations,wherein numbering is according to the EU index of Kabat; (iii) a humanIgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236mutations, wherein numbering is according to the EU index of Kabat; or(iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A,delG236, and N297A mutations, wherein numbering is according to the EUindex of Kabat. In some embodiments, the polypeptide (e.g., fusionpolypeptide) administered to the individual comprises the amino acidsequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, thepolypeptide (e.g., fusion polypeptide) forms a homodimer. In someembodiments, the polypeptide (e.g., fusion polypeptide), and theanti-TROP2 antibody are administered simultaneously, concurrently, orsequentially. In some embodiments, the cancer is solid tumor, gastriccancer, nasopharyngeal cancer, gallbladder cancer, cervical cancer,extranodal NK/T cell lymphoma, lung cancer, laryngeal squamous cellcancer, colon cancer, Hilar Cholangiocarcinoma, pancreatic cancer,squamous cell carcinoma of the oral cavity, endometrioid endometrialcarcinoma, or ovarian carcinoma. In some embodiments, the cancer ischaracterized by the overexpression of TROP2. In some embodiments, thecancer is not characterized by the overexpression of TROP2.

Methods of Increasing Phagocytosis of a Target Cell

In some embodiments, provided is a method of increasing phagocytosis ofa target cell (e.g., a cancer cell) that comprises contacting the targetcell with (a) an agent that blocks the interaction between CD47 (e.g.,hCD47) and SIRPα (e.g., hSIRPα) and (b) an anti-TROP2 antibody. In someembodiments, the anti-TROP2 antibody is RS7, which is described in U.S.Pat. No. 10,179,171, the contents of which are incorporated herein intheir entirety. In some embodiments, the anti-TROP2 antibody isconjugated to a drug (i.e., an antibody-drug conjugate or “ADC”). Insome embodiments, the anti-TROP2 ADC is Sacituzumab govitecan (alsoknown as hRS7-SN38 or IMMU-132), which is described in US 2017/0281791,the contents of which are incorporated herein by reference in theirentirety. In some embodiments, the agent is a polypeptide (e.g., fusionpolypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1domain variant described herein) and an Fc domain variant (e.g., an Fcdomain variant described herein). In some embodiments, the polypeptide(e.g., fusion polypeptide) comprises a SIRPα D1 domain variant thatcomprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. Insome embodiments, the Fc domain variant is (i) a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat. Insome embodiments, the polypeptide (e.g., fusion polypeptide)administered to the individual comprises the amino acid sequence of SEQID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide(e.g., fusion polypeptide) forms a homodimer. In some embodiments, thetarget cell is a cancer cell. In some embodiments, the cancer cell is asolid tumor cell, a gastric cancer cell, a nasopharyngeal cancer cell, agallbladder cancer cell, a cervical cancer cell, an extranodal NK/T celllymphoma cell, a lung cancer cell, a laryngeal squamous cell cancercell, a colon cancer cell, a Hilar Cholangiocarcinoma cell, a pancreaticcancer cell, a squamous cell carcinoma cell of the oral cavity, anendometrioid endometrial carcinoma cell, or an ovarian carcinoma cell.

In some embodiments, provided is a method of increasing phagocytosis ofa target cell comprising contacting the target cell with (a) an agentthat blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g.,hSIRPα) and (b) a second agent that is capable of enhancingphagocytosis. In some embodiments, the agent that blocks the interactionbetween CD47 and SIRPα is a polypeptide (e.g., fusion polypeptide)comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variantdescribed herein) and an Fc domain variant (e.g., an Fc domain variantdescribed herein). In some embodiments, the polypeptide (e.g., fusionpolypeptide) comprises a SIRPα D1 domain variant that comprises theamino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In someembodiments, the Fc domain variant is (i) a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat. Insome embodiments, the polypeptide (e.g., fusion polypeptide)administered to the individual comprises the amino acid sequence of SEQID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide(e.g., fusion polypeptide) forms a homodimer. In some embodiments, thesecond agent enhances phagocytosis, e.g., by blocking “don't eat me”signals. Exemplary agents include, but are not limited to, e.g., ananti-LILRB2 antibody, an anti-LILRB1 antibody, an anti-SIGLEC-10antibody, an anti-CD24 antibody, an anti-SIRPα antibody, an anti-PD1antibody (e.g., an anti PD1 antagonist antibody), and an anti-PD-L1antibody (e.g., an anti PD-L1 antagonist antibody). In some embodiments,the second agent enhances phagocytosis, e.g., by enhancing “eat me”signals. Exemplary agents include, but are not limited to, e.g., BTKactivators, TLR agonists, agents that promote the interaction betweenMac-1 and SLAMF7, and agents that agents that promote the interactionbetween calreticulin and LRP1. Additional exemplary agents that enhancephagocytosis include, but are not limited to, e.g., agents that modulatepodosome adhesions, agents that modulate the expression level of laminA, activators of the SHP-1 phosphatase activity, and activators ofmyosin Ha assembly. In some embodiments, the method comprises contactingthe target cell with (a) the polypeptide (e.g., fusion polypeptide)comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variantdescribed herein) and an Fc domain variant (e.g., an Fc domain variantdescribed herein) and (b) and an anti-LILBR2 antibody, an anti-CD24antibody, or an anti-SIGLEC-10 antibody. In some embodiments, the methodcomprise contacting the target cell with (a) the fusion polypeptide and(b) a BTK activator. In some embodiments, the method comprisescontacting the target cell with (a) the fusion polypeptide and (b) a TLRagonist.

In some embodiments, the method comprises contacting the target cellwith (a) the polypeptide (e.g., fusion polypeptide) comprising a SIRPαD1 domain variant (e.g., a SIRPα D1 domain variant described herein) andan Fc domain variant (e.g., an Fc domain variant described herein) and(b) two or more agents that are capable of enhancing phagocytosis (e.g.,including, but not limited to, two or more agents described herein). Insome embodiments, the method comprises contacting the target cell with(a) the polypeptide (e.g., fusion polypeptide) comprising a SIRPα D1domain variant (e.g., a SIRPα D1 domain variant described herein) and anFc domain variant (e.g., an Fc domain variant described herein), (b) andan anti-LILBR2 antibody, an anti-CD24 antibody, or an anti-SIGLEC-10antibody, and (c) an anti-PD1 antibody (e.g., an anti-PD-1 antagonistantibody) or an anti-PD-L1 antibody (e.g., an anti-PD-L1 antagonistantibody). In some embodiments, the method comprises contacting thetarget cell with (a) the fusion polypeptide, (b) an anti-LILBR2antibody, and (c) an anti-PD1 antibody (e.g., anti-PD-1 antagonistantibody). In some embodiments, the method comprises contacting thetarget cell with (a) the fusion polypeptide, (b) an anti-LILBR2antibody, and (c) an anti-PD-L1 antibody (e.g. an anti-PD-L1 antagonistantibody).

In some embodiments, the contacting is performed in vitro. In someembodiments, the contacting is performed in vivo. In some embodiments,the target cell is a cancer cell. In some embodiments, contacting thetarget cell with (a) the polypeptide comprising a SIRPα D1 domainvariant (e.g., a SIRPα D1 domain variant described herein) and an Fcdomain variant (e.g., an Fc domain variant described herein) and (b) oneor more agents that are capable of enhancing phagocytosis increasesphagocytosis of target cells by at least any one of 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 99%, or more than 99% as compared contacting the target cell withone or more agents that are capable of enhancing phagocytosis (i.e., inthe absence of the polypeptide comprising a SIRPα D1 domain variant(e.g., a SIRPα D1 domain variant described herein) and an Fc domainvariant (e.g., an Fc domain variant described herein)).

Kits and Articles of Manufacture

In another embodiment of the invention, an article of manufacture or akit is provided comprising a polypeptide (e.g., a fusion polypeptidedescribed herein) comprising a SIRPα D1 domain variant and an Fc domainvariant. In some embodiments, the SIRPα D1 domain variant comprises theamino acid sequence selected from the group consisting of: SEQ ID NO: 81and SEQ ID NO: 85. In some embodiments, the Fc domain variant is (i) ahuman IgG1 Fc region comprising L234A, L235A, G237A, and N297Amutations, wherein numbering is according to the EU index of Kabat; (ii)a human IgG2 Fc region comprising A330S, P331S, and N297A mutations,wherein numbering is according to the EU index of Kabat; (iii) a humanIgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236mutations, wherein numbering is according to the EU index of Kabat; or(iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A,delG236, and N297A mutations, wherein numbering is according to the EUindex of Kabat. In some embodiments, the Fc domain variant comprises theamino acid sequence of SEQ ID NO: 91. In some embodiments thepolypeptide comprises the amino acid sequence of SEQ ID NO: 135 or SEQID NO: 136. In some embodiments, the kit or article of manufacture isfor use according to a method of treatment provided herein.

In some embodiments, the kit or article of manufacture further comprisesa BCL2 inhibitor. In some embodiments, the BCL2 inhibitor is venetoclax.In some embodiments, the kit comprises a package insert or label withinstructions for using the polypeptide (e.g., fusion polypeptide) incombination with the BCL2 inhibitor (e.g., venetoclax) to treat or delayprogression of cancer (e.g., leukemia, including, but not limited toacute or chronic lymphoblastic leukemia, acute lymphocytic leukemia(ALL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma(SLL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS),chronic myeloid leukemia (CIVIL), hairy cell leukemia, Chronicmyelomonocytic leukemia (CMML), Juvenile myelomonocytic leukemia (JMML),Large granular lymphocytic (LGL) leukemia, blastic plasmacytoiddendritic cell neoplasm (BPDCN), B-cell prolymphocytic leukemia (B-PLL),T-cell prolymphocytic leukemia (T-PLL), Multiple Myeloma (MM), andNon-Hodgkin Lymphomas (such as diffuse large B-cell lymphoma (DLBCL),Burkitt lymphoma, mantle cell lymphoma (MCL), Peripheral T-cell lymphoma(PTCL), Lymphoplasmacytic lymphoma, Waldenström macroglobulinemia,marginal zone lymphoma (MZL) and follicular lymphoma (FL)) in anindividual (such as a human individual).

In some embodiments, the kit or article of manufacture further comprisesa platinum-based chemotherapy agent. In some embodiments, theplatinum-based chemotherapy agent is carboplatin, cisplatin,oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin,picoplatin, or satraplatin. In some embodiments, the kit comprises apackage insert or label with instructions for using the polypeptide(e.g., fusion polypeptide) in combination with the platinum-basedchemotherapy agent (e.g., cisplatin) to treat or delay progression ofsolid tumor (e.g., colon cancer, colon carcinoma, lung cancer, head andneck cancer, esophageal cancer, breast cancer, bladder cancer, ovariancancer, cervical cancer, testicular cancer, endometrial cancer, livercancer, gastric cancer, brain tumor, mesothelioma, or neuroblastoma) inan individual (such as a human individual).

In some embodiments, the kit or article of manufacture further comprisesan anti-HER2 antibody (e.g., trastuzumab), and a PD-L1 inhibitor (e.g.,an anti-PD-L1 antibody such as atezolizumab, avelumab, or durvalumab. Insome embodiments, the kit comprises a package insert or label withinstructions for using the polypeptide (e.g., fusion polypeptide) incombination with the anti-HER2 antibody (e.g., trastuzumab), the PD-L1inhibitor (e.g., atezolizumab, avelumab, or durvalumab) to treat ordelay progression of cancer (e.g., solid tumor) in an individual (suchas a human individual). In some embodiments, the cancer (e.g., solidtumor) is colon cancer, lung cancer, head and neck cancer, esophagealcancer, breast cancer, bladder cancer, ovarian cancer, cervical cancer,testicular cancer, endometrial cancer, liver cancer, gastric cancer,gastroesophageal junction cancer, brain tumor, mesothelioma, orneuroblastoma. In some embodiments, the cancer (e.g., solid tumor) isHER2⁺ cancer. In some embodiments, the cancer is colon cancer (e.g.,HER2⁺ colon cancer).

In some embodiments, the kit or article of manufacture further comprisesan anti-HER2 antibody (e.g., trastuzumab), an anti-VEGFR2 antibody(e.g., ramucirumab), and paclitaxel. In some embodiments, the kitcomprises a package insert or label with instructions for using thepolypeptide (e.g., fusion polypeptide) in combination with the anti-HER2antibody (e.g., trastuzumab), the anti-VEGFR2 antibody (e.g.,ramucirumab), and the paclitaxel to treat or delay progression ofgastric cancer or gastroesophageal junction (GEJ) cancer in anindividual (such as a human individual), e.g., according to a methoddescribed herein.

In some embodiments, the kit or article of manufacture further comprisesan anti-HER2 antibody (e.g., trastuzumab), a PD-1 inhibitor (e.g., ananti-PD-1 antibody such as pembrolizumab), 5-fluorouracil, and aplatinum-based agent (e.g., cisplatin or carboplatin). In someembodiments, the kit comprises a package insert or label withinstructions for using the polypeptide (e.g., fusion polypeptide) incombination with the anti-HER2 antibody (e.g., trastuzumab), the PD-1inhibitor (e.g., pembrolizumab), the 5-fluorouracil, and theplatinum-based agent (e.g., cisplatin or carboplatin) to treat or delayprogression of gastric cancer or gastroesophageal junction (GEJ) cancerin an individual (such as a human individual). In some embodiments, thekit or article of manufacture further comprises an anti-HER2 antibody(e.g., trastuzumab), a PD-1 inhibitor (e.g., an anti-PD-1 antibody suchas pembrolizumab), capecitabine, and a platinum-based agent (e.g.,cisplatin or carboplatin). In some embodiments, the kit comprises apackage insert or label with instructions for using the polypeptide(e.g., fusion polypeptide) in combination with the anti-HER2 antibody(e.g., trastuzumab), the PD-1 inhibitor (e.g., pembrolizumab), thecapecitabine, and the platinum-based agent (e.g., cisplatin orcarboplatin) to treat or delay progression of gastric cancer orgastroesophageal junction (GEJ) cancer in an individual (such as a humanindividual).

In some embodiments, the kit or article of manufacture further comprisesa PD-1 inhibitor (e.g., an anti-PD-1 antibody such as pembrolizumab,nivolumab, pidilizumab, cemiplimab, or BMS936559), an antimetabolite(e.g., 5-fluorouracil, 6-mercaptopurine, capecitabine, cytarabine,floxuridine, fludarabine, gemcitabine, hydroxycarbamide, methotrexate,pemetrexed, phototrexate) and a platinum-based agent (e.g., cisplatin,carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate,phenanthriplatin, picoplatin, or satraplatin). In some embodiments, thekit comprises a package insert or label with instructions for using thepolypeptide (e.g., fusion polypeptide) in combination with the PD-1inhibitor (e.g., pembrolizumab, nivolumab, pidilizumab, cemiplimab, orBMS936559), the antimetabolite (e.g., 5-fluorouracil, 6-mercaptopurine,capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine,hydroxycarbamide, methotrexate, pemetrexed, phototrexate), and theplatinum-based agent (e.g., cisplatin, carboplatin, oxaliplatin,nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, orsatraplatin) to treat or delay progression of head and neck cancer(e.g., head and neck squamous cell carcinoma) in an individual (such asa human individual), e.g., according to a method provided herein.

In some embodiments, the kit or article of manufacture further comprisesa therapeutic anti-TROP2 antibody. In some embodiments, the anti-TROP2antibody is RS7 (see, e.g., U.S. Pat. No. 10,179,171) or sacituzumabgovitecan. In some embodiments, the kit comprises a package insert orlabel with instructions for using the polypeptide (e.g., fusionpolypeptide) in combination with anti-TROP2 antibody (e.g., cisplatin)to treat or delay progression of a TROP2⁺ cancer (e.g., solid tumor,gastric cancer, nasopharyngeal cancer, gallbladder cancer, cervicalcancer, extranodal NK/T cell lymphoma, lung cancer, laryngeal squamouscell cancer, colon cancer, Hilar Cholangiocarcinoma, pancreatic cancer,squamous cell carcinoma of the oral cavity, endometrioid endometrialcarcinoma, or ovarian carcinoma) in an individual (such as a humanindividual).

In some embodiments, the polypeptide (e.g., fusion polypeptide) and theone or more additional anti-cancer agents (e.g., as outlined in theembodiments above) are provided together in the kit. In someembodiments, the polypeptide (e.g., fusion polypeptide) and the one ormore additional anti-cancer agents are provided in the same container orseparate containers. Suitable containers include, for example, bottles,vials, bags and syringes. The container may be formed from a variety ofmaterials such as glass, plastic (such as polyvinyl chloride orpolyolefin), or metal alloy (such as stainless steel or hastelloy). Insome embodiments, the container holds the formulation and the label on,or associated with, the container may indicate directions for use. Thearticle of manufacture or kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use. In some embodiments, the article of manufacturefurther includes one or more of another agent (e.g., a chemotherapeuticagent, an anti-neoplastic agent, a therapeutic antibody, etc.). Suitablecontainers for the one or more agents include, for example, bottles,vials, bags and syringes.

The specification is considered to be sufficient to enable one skilledin the art to practice the invention. Various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

EXAMPLES

The present disclosure will be more fully understood by reference to thefollowing examples. The examples should not, however, be construed aslimiting the scope of the present disclosure. It is understood that theexamples and embodiments described herein are for illustrative purposesonly and that various modifications or changes in light thereof will besuggested to persons skilled in the art and are to be included withinthe spirit and purview of this application and scope of the appendedclaims

Example 1A: Anti-Tumor Activity of Drug a in Combination with Venetoclaxin an Acute Leukemia Model

In this Example, the anti-tumor activity of Drug A, i.e., an exemplarypolypeptide comprising a SIRPα d1 domain variant and an Fc variant, incombination with venetoclax was assessed in a RS4; 11 xenograft model.

Materials and Methods

RS4; 11 Xenograft Model

The RS4; 11 cells (described in Stong et al. (1985) Blood. 65(1): 21-31)was injected into the right flank of NOD-SCID female mice at aconcentration of 5×10⁶ cells per mouse using a 1:1 matrigel (Corning)and RPMI 1640 ratio. Tumors were monitored until the average size of alltumors reached 190 mm³. Mice were randomized into PBS control,Venetoclax (Selleckchem), Drug A, and Venetoclax/Drug A combinationcohorts, with 10 mice per cohort. The formulation for Venetoclax was aratio of DMSO:ethanol:Cremophor EL:dextrose 5% in water (D5W) was2.5:5:10:20:67.5, by volume. Venetoclax-treated mice were dosed with 250μg of Venetoclax by oral gavage 2 times total, 3 days apart. DrugA-treated mice were dosed IP at 10 mg/kg, 4 times total, 3-4 days apart.Venetoclax/Drug A-treated mice were dosed with 250 μg of Venetoclax byoral gavage 2 times total, 3 days apart, and with Drug A 1 day postVenetoclax dosage at 10 mg/kg, 4 times total, 3-4 days apart. Tumorswere measured in two dimensions with calipers, and tumor volume wascalculated as: length×width×width×0.5, where length was the larger ofthe two measurements.

Results

Single agent venetoclax inhibited tumor growth (see FIG. 1A), whereassingle agent Drug A did not have an appreciable effect on tumor growth.The combination of venetoclax and Drug A inhibited tumor growth to agreater degree than venetoclax alone (see FIG. 1A). On day 41, 1 out of10 of the mice treated with Venetoclax only was tumor-free (“TF”),whereas 6 out of 10 mice treated with the Venetoclax/Drug A combinationwere TF (FIG. 1A).

The Venetoclax-treated mice (n=10) were then split into two groups(n=5/group) and either (a) re-treated with single agent Venetoclax onDay 45, or (b) treated with Venetoclax (administered on Day 45) incombination with Drug A (administered on Day 46). As shown in FIG. 1B,in mice who had received prior Venetoclax, treatment with Venetoclax incombination with Drug A inhibited tumor growth to a greater extent thatre-treatment with Venetoclax alone. The average tumor volume at Day 65of mice re-treated with Venetoclax was about 1685 mm³, whereas theaverage tumor volume at Day 65 of mice treated with the combination wasabout 970 mm³. The mouse that demonstrated tumor regression when treatedwith single-agent Venetoclax received single agent Venetoclax on Day 45.Notably, tumor regrowth was observed in this mouse.

Example 1B: Effect of Drug a in Combination with Venetoclax onPhagocytosis by Macrophages in an In Vitro Model

In this Example, the effects of Drug A alone, venetoclax alone, and DrugA in combination with venetoclax on the phagocytosis of HL60 and OCIAML3human acute myeloid leukemia cells by macrophages were assessed in an invitro assay.

Materials and Methods

Derivation and Culture of Human Monocyte-Derived Macrophages forPhagocytosis

CD14⁺ monocytes were purified by negative selection using the ClassicalMonocytes Isolation Kit, human (Miltenyi Biotec) and LS columns(Miltenyi Biotec) according to the manufacturer's protocol. CD14⁺monocytes were seeded into 150 mm tissue culture dishes (Corning) at 6million cells per dish in 25 mL medium comprised of RPMI complete media,supplemented with 50 ng/mL M-CSF (Miltenyi Biotec), 10% human FBS serum(Thermo Fisher Scientific), 1% penicillin/streptomycin, and 1% GlutaMAX.Cells were cultured for seven to eleven days.

In Vitro Phagocytosis Assays

HL60 and OCI-AML3 cells were washed once in PBS and labeled with theCelltrace CFSE Cell Proliferation kit (Thermo Fisher Scientific) insuspension with 300 nM CFSE (carboxyfluorescein succinimidyl ester)according to the manufacturer's instructions and resuspended in RPMIcomplete media. Target cells were incubated overnight with two-foldserial dilutions of venetoclax between 39 nM to 2.5 μM in RPMI completemedia. Prior to incubation with macrophages, cells were resuspended inRPMI. Macrophages were detached from culture plates by washing once withPBS and incubation in TrypLE Select for 20 minutes at 37° C. Cells wereremoved with a cell scraper (Corning), washed in PBS, and resuspended inRPMI.

CFSE-labeled target cells treated with venetoclax for 48 hours were spunand added to ultra-low attachment U-bottom 96 well plates (Corning) at100,000 cells per well. Drug A was then added. Plates were incubated 30minutes at 37° C. in a humidified incubator with 5% carbon dioxide, then50,000 macrophages were added. Plates were incubated two hours at 37° C.in a humidified incubator with 5% carbon dioxide. Cells were pelleted bycentrifugation for five minutes at 400×g and stained at 4° C. for 30minutes in Fixable Viability Dye eFluor 780 (ebioscience) diluted 1:4000in PBS. Cells were washed in FACS buffer (PBS with 0.5% BSA) and stainedat 4° C. for 45 minutes in FACS buffer containing human FcR BlockingReagent (Miltenyi Biotec), BV421 anti-CD33 (Biolegend), APC anti-CD14(Biolegend) and PE-Cyanine7 anti-CD11b (Invitrogen). Cells were washedtwice in FACS buffer and fixed overnight at 4 degrees C. in 0.5%paraformaldehyde diluted in PBS. Cells were analyzed on a FACS Canto II(BD Biosciences), with subsequent data analysis by Flowjo 10.6.1 (BectonDickinson & Company). Dead cells were excluded by gating on thee780-negative population. Macrophages were identified as cell positivefor the lineage markers CD33, CD11b and CD14. Of this population,macrophages that had phagocytosed tumor cells were identified as cellspositive for CFSE.

Results

Briefly HL60 cells and OCI-AML3 cells (i.e., “target cells”) werelabeled with CFSE (carboxyfluorescein succinimidyl ester) and treatedwith venetoclax for 48 hours. The target cells were then spun and addedto wells of 96 well plates at 100,000 cells per well. Drug A was thenadded. Untreated control target cells, as well as control target cellsthat treated only with venetoclax or only with Drug A, were prepared inparallel. Macrophages were added to the wells, and the plates wereincubated two hours at 37° C. Macrophage cells were pelleted, stained,and analyzed via flow cytometry. Dead cells were excluded by gating onthe e780-negative population. Macrophages were identified as cellpositive for the lineage markers CD33, CD11b and CD14. Of thispopulation, macrophages that had phagocytosed tumor cells wereidentified as cells positive for CFSE.

As shown in FIG. 5A, venetoclax as a single agent stimulatedmacrophage-mediated phagocytosis of HL60 cells, whereas Drug A as asingle agent had little effect phagocytosis. (Compare Drug A treatedcells to that of untreated cells.) The combination of 20 nM Drug A and125 nM venetoclax stimulated phagocytosis of HL60 cells by macrophagesto a greater degree than either Drug A alone or venetoclax alone.Similar results were observed using 20 nM Drug A and 1 μM venetoclax inOCI-AML3 cells. See FIG. 5B.

Example 2: Anti-Tumor Activity of Drug a in Combination with Cisplatinin a Colon Carcinoma Model

In this Example, the anti-tumor activity of Drug A in combination withcisplatin was assessed in a CT26 syngeneic mouse colon carcinoma model.See, e.g., Mosely et al. (2016) “Rational Selection of SyngeneicPreclinical Tumor Models for Immunotherapeutic Drug Discovery.” CancerImmunol Res. 5(1): 29-41.

Materials and Methods

CT26 Syngeneic Model

The CT26 cells (see Wang et al. (1995) J Immunol. 154:4685-4692) wereinjected into the right flank of BALB/c female mice at a concentrationof 5×10⁵ cells per mouse in RPMI 1640. Tumors were monitored until theaverage size of all tumors reached 65-70 mm³. Mice were randomized intoPBS control, cisplatin (Selleckchem), Drug A, and cisplatin/Drug Acombination cohorts, with n=5-10 per cohort. Drug A was administeredintraperitoneally (IP) at twice at a dose of 30 mg/kg (“mpk”). The two30 mpk doses were given 10 days apart. Cisplatin was administered IPaccording to one of two regimens: once at a dose of 10 mpk or twice at adose of 5 mpk. The two 5 mpk cisplatin doses were given 10 days apart.Mice receiving both cisplatin and Drug were administered with cisplatin(IP) according to one of the regimens described above, and with Drug Aas described above. In mice receiving combination treatment, Drug A wasadministered one day after cisplatin. Tumors were measured in twodimensions with calipers, and tumor volume was calculated as:length×width×width×0.5, where length was the larger of the twomeasurements.

Results

As shown in FIG. 2A, at day 20, tumor growth in mice treated with singleagent cisplatin (two 5 mpk doses, each given 10 days apart) wasinhibited somewhat, whereas Drug A did not have an appreciable effect oftumor growth. Treatment with cisplatin in combination with Drug Adelayed CT26 tumor growth in mice to a greater degree than either drugalone. Further, as shown in FIG. 2B, mice treated with cisplatin incombination with Drug A gained more weight during the course oftreatment than mice given cisplatin alone. Additionally, only 10% of themice in each of the PBS control, cisplatin, and Drug A treatment groupsfound to have a tumor volume<500 mm³, whereas 33% of the mice in thecisplatin+Drug A treatment groups had tumors<500 mm³ in volume.

Similar results were observed in the groups treated with a single 10 mpkdose of cisplatin, either alone or in combination with Drug A. Treatmentwith cisplatin in combination with Drug A delayed CT26 tumor growth inmice to a greater degree than either drug alone. See FIG. 2C. See Micetreated with cisplatin alone or in combination with Drug A showedsimilar body weight change (*p<0.0106 and **p<0.0021, two-tailed t-testwas performed on Days 24 and 27, respectively, between cisplatin andDrug A+cisplatin treated groups). See FIG. 2D.

Example 3: Phagocytic Activity of Drug a in Combination with anAnti-TROP2 Antibody

DLD-1 cells were washed twice with 20 ml PBS and incubated in 10 mlTRYPLE™ Select (Gibco) cell-dissociation enzymes for 10 minutes at 37°C. in order to detach the cells from the culture plates. The detachedcells were then centrifuged, washed in PBS, and resuspended in medium.Cells were labeled with the fluorescent label provided with theCELLTRACE™ CFSE Cell Proliferation kit (Thermo Fisher) according to themanufacturer's instructions and resuspended in IMDM (Iscove's ModifiedDulbecco Medium). Macrophages were detached from culture plates bywashing twice with 20 ml PBS and incubation in 10 ml TRYPLE™ Select(Gibco) cell-dissociation enzymes for 20 minutes at 37° C. Cells wereremoved with a cell scraper (Corning), washed in PBS, and resuspended inIMDM.

Phagocytosis assays were assembled in ultra-low attachment U-bottom 96well plates (Corning) containing 100,000 DLD-1, 50,000 macrophages,five-fold serial dilutions of Drug A or negative control antibody from100 nM to 6.4 pM, and anti-TROP2 antibody at 0.01 μg/ml. The plates wereincubated two hours at 37° C. in a humidified incubator with 5 percentcarbon dioxide. Cells were then pelleted by centrifugation for fiveminutes at 400×g and washed in 250 μl FACS buffer. Macrophages werestained on ice for 15 minutes in 50 μl FACS buffer containing 10 μlhuman FcR Blocking Reagent (Miltenyi Biotec), 0.50 anti-CD33 Abconjugated to BV421 label (Biolegend), and 0.5 μl anti-CD206 conjugatedto Allophycocyanin-Cy7 label (Biolegend). Next, the cells were washed in200 μl FACS buffer, washed in 250 μl PBS, and stained on ice for 30minutes in 50 μl Fixable Viability Dye EFLUOR™ 506 (ebioscience)viability dye diluted 1:1000 in PBS. Cells were then washed twice in2500 FACS buffer and fixed overnight in 0.5% paraformaldehyde. The fixedcells were analyzed on a FACS CANTO II™ (BD Biosciences)fluorescence-activated cell sorting analyzer, with subsequent dataanalysis by FlowJo 10.7 (Treestar) flow cytometry software. Dead cellswere excluded by gating on the e506-negative population. Macrophagesthat had phagocytosed tumor cells were identified as cells positive forCD33, CD206, and CFSE (i.e., carboxyfluorescein succinimidyl ester).

Enhanced phagocytosis of CFSE-labeled DLD-1 tumor cells by humanmonocyte-derived macrophages in the presence of Drug A in combination ofanti-Trop2. In FIG. 3, the percent of macrophages that phagocytosedCFSE-labeled tumor cells is indicated on the y-axis. Macrophages wereincubated with the indicated concentration of Drug A and 10 ng/mLanti-TROP2 antibody. Cells were also incubated with 10 ng/mL anti-TROP2antibody alone, with negative control human IgG antibody in combinationwith anti-TROP2 antibody, and in media only. Phagocytosis ofCFSE-labeled DLD-1 tumor cells by human monocyte-derived macrophages wasenhanced in the presence of Drug A in combination of anti-TROP2antibody. See FIG. 3.

Example 4: Anti-Tumor Activity of Drug a in Combination with Trastuzumaband an Anti-PD1 Antibody in a Colon Cancer Model

MC38 m/h HER2 cells were generated by infecting MC38 murine colonadenocarcinoma cells with a lentivirus vector encoding a chimera ofmouse and human HER2 transmembrane and extracellular domains. MC38 m/hHER2 cells were maintained in DMEM (Thermo Fisher Scientific 11965092)supplemented with 10% FBS, 1% Penicillin-Streptomycin, 1% GlutaMAX and 1mM Sodium Pyruvate (Thermo Fisher Scientific 11360070) at 37° C., 5% CO2incubator. All tissue culture was performed under aseptic conditions.

Prior to implantation, a master cell bank of each cell line wasgenerated to assure that cells used in subsequent experiments were ofthe same passage number. Cells were harvested and washed two times in 50mL cold PBS (Life Technologies 10010072). After the final wash, cellswere resuspended in PBS or RPMI at 5×10⁶ cells/mL for MC38 m/h HER2 cellline. 100 μL of cell suspension were subcutaneously injected into theright flank of C57BL/6 mice for MC38 m/h HER2. When tumor size reachedan average of 65-69 mm³ for MC38 m/h HER2 tumors, the animals wererandomized into 8 groups of 10 mice. Each group was assigned to atreatment group outlined in Table A:

TABLE A Treatment Groups—MC38 m/h Tumor Model Treatment Group Dosingregimen Drug A Drug A (IP): 30 mg/kg, 2q10d (single agent) anti-PDL1antibody anti-PDL antibody (IP): 1 mg/kg, 3q5d (single agent)Trastuzumab trastuzumab (IP): 30 mg/kg, 3q5d (single agent) Drug A +anti-PDL1 Drug A (IP): 30 mg/kg, 2q10d antibody doublet anti-PDL1antibody (IP): 1 mg/kg, 3q5d Drug A + trastuzumab Drug A (IP): 30 mg/kg,2q10d doublet trastuzumab (IP): 30 mg/kg, 3q5d anti-PDL1 antibody +anti-PDL1 antibody (IP): 1 mg/kg, 3q5d trastuzumab doublet trastuzumab(IP): 30 mg/kg, 3q5d Drug A + anti-PDL1 Drug A (IP): 30 mg/kg, 2q10dantibody + trastuzumab anti-PDL1 antibody (IP): 1 mg/kg, 3q5d PBS(control)

Tumor volume (mm³) using Mitutoyo Digital Caliper (Mitutoyo America,Aurora, Ill.) and body weights were recorded two or three times perweek. Mice exceeding tumor volume of 2000 mm³ or loss of 20% body weightwere euthanized according to IACUC guidelines. Tumor volume iscalculated ([length×{width×width}]×0.5=volume in mm³). Statisticalanalysis and p values were calculated using GraphPad Prism Software.

Chimeric m/h HER2, with the extracellular domain from human HER2 andintracellular domain from mouse HER2, was expressed on MC38 colon cellsto permit evaluation of trastuzumab's activity against MC38 murinetumors. As shown in FIG. 5, Monotherapy with trastuzumab had no effecton tumor growth, while Drug A monotherapy and anti-PD-L1 antibodymonotherapy each had a moderate effect on tumor growth. Treatment withthe Drug A+anti-PD-L1 antibody doublet or the trastuzumab+anti-PD-L1antibody doublet showed improved tumor growth inhibition as compared tomonotherapy alone. Treatment with the Drug A+anti-PD-L1+trastuzumabtriple combination showed improved tumor inhibition when compared toeach doublet. The effect in reducing tumor growth of the triplecombination as compared to the Drug A+anti-PD-L1 antibody doublet or thetrastuzumab+anti-PD-L1 antibody doublet was most evident on days 19 and22, which were 3-6 days post last dose. By day 26, triple combinationwas minimally better at reducing tumor growth as compared to the DrugA+anti-PD-L1 antibody doublet or the trastuzumab+anti-PD-L1 antibodydoublet. No adverse effects were observed in any of the treatmentcohorts in the MC38 m/h HER2 colon tumor models.

Example 5A: Exemplary Clinical Trials to Assess the Anti-Tumor Activityof Drug a Combination Therapies in Human Patients

Gastric or Gastroesophageal Junction (GEJ) Adenocarcinoma

A clinical trial is performed to assess the safety, tolerability, andefficacy of the combination of Drug A, trastuzumab, ramucirumab, andpaclitaxel in patients with HER2⁺ overexpressing advanced or metastaticgastric or GEJ adenocarcinoma that has progressed during or after priortherapy with trastuzumab and fluoropyrimidine-containing chemotherapy(e.g., fluorouracil); during or after prior therapy with trastuzumab andplatinum-containing chemotherapy; or during or after prior therapy withtrastuzumab, fluoropyrimidine-containing chemotherapy (e.g.,fluorouracil), and platinum-containing chemotherapy. The patientsenrolled in the trial are suitable for treatment with trastuzumab. Thepatients have not received prior therapy with an anti-CD47 agent or ananti-SIRPα agent.

A clinical trial is performed to assess the safety, tolerability, andefficacy of the combination of Drug A, pembrolizumab, cisplatin, andeither 5-fluorouracil or capecitabine in patients with gastric or GEJadenocarcinoma (e.g., HER2⁺ overexpressing gastric or GEJadenocarcinoma). The patients enrolled in the trial have not receivedprior therapy with an anti-CD47 agent or an anti-SIRPα agent. Patientshave adequate organ function and hemoglobin is greater or equal to 9g/dL.

Head and Neck Squamous Cell Carcinoma (HNSCC)

A clinical trial is performed to assess the safety, tolerability, andefficacy of the combination of Drug A, pembrolizumab, 5-fluorouracil,and either carboplatin or cisplatin in patients with metastatic or withunresectable, recurrent HNSCC who have not yet been treated for theiradvanced disease.

Example 5B: Preliminary Safety Results from the Exemplary ClinicalTrials Described in Example 5A

One patient with untreated advanced head and neck squamous cellcarcinoma received treatment with Drug A (10 mg/kg IV QW), pembrolizumab(200 mg IV Q3W), 5-fluorouracil (1,000 mg/m² per day on days 1, 2, 3, 4Q3W×6), and carboplatin (AUC=5 mg/ml/min on Day 1, Q3W×6). (In expansionstudies, cisplatin (100 mg/m² Q3Wx 6) or carboplatin (AUC=5 mg/ml/min onDay 1, Q3W×6) is administered in combination with Drug A, pembrolizumab,and fluorouracil. Patients who receive carboplatin continue to receivecarboplatin for the duration of the expansion studies; patients whoreceive cisplatin continue to receive cisplatin for the duration of theexpansion studies). Three patients with HER2-positivegastric/gastroesophageal cancer who progressed on prior treatment(s)with trastuzumab, fluorouracil, and a platinum agent received treatmentwith Drug A (10 mg/kg IV QW), trastuzumab (8 mg/kg IV for the initialdose, followed by 6 mg/kg Q3W), ramucirumab (8 mg/kg on Days 1 and 15Q4W), and paclitaxel (80 mg/m2 on Days 1, 8, and 15 Q4W). Threeadditional patients with HER2-positive gastric/gastroesophageal cancerwho progressed on prior treatment(s) with trastuzumab, fluorouracil, anda platinum agent received treatment with Drug A (15 mg/kg IV QW),trastuzumab (8 mg/kg IV for the initial dose, followed by 6 mg/kg Q3W),ramucirumab (8 mg/kg on Days 1 and 15 Q4W), and paclitaxel (80 mg/m2 onDays 1, 8, and 15 Q4W).

Initial results suggest that Drug A, when administered a dose of 10mg/kg or 15 mg/kg QW in the combination regimens discussed above, iswell tolerated with no dose-limiting toxicities to date. Three patients(50%) administered with Drug A+trastuzumab+ramucirumab+paclitaxel and nopatient (0%) administered with DrugA+pembrolizumab+fluorouracil+carboplatin experienced treatment-relatedadverse events (TRAEs). There were no dose limiting toxicities inpatients receiving Drug A+pembrolizumab+fluorouracil+carboplatin or DrugA+trastuzumab+ramucirumab+paclitaxel. There were also no treatmentrelated adverse events (TRAE) that occurred in two or more patients inthe following 3 cohorts:

Drug A (10 mg/kg QW)+pembrolizumab+fluorouracil+carboplatin (N=1)

Drug A (10 mg/kg QW)+trastuzumab+ramucirumab+paclitaxel (N=3)

Drug A (15 mg/kg QW)+trastuzumab+ramucirumab+paclitaxel (N=3)

And finally, there were also no grade 3 or above Treatment relatedadverse events (TRAE≥Grade 3) reported in patients treated with DrugA+pembrolizumab+fluorouracil+carboplatin or DrugA+trastuzumab+ramucirumab+paclitaxel.

Example 5C: Preliminary Efficacy Results from the Exemplary ClinicalTrials Described in Example 5A

The patient with untreated advanced head and neck squamous cellcarcinoma who received treatment with Drug A, pembrolizumab,5-fluorouracil, and a platinum agent at the dosages and administrationschedule described in Example 5B achieved partial response (PR) based oninvestigator-assessed response using RECIST v1.1 criteria.

Among the three patients with HER2-positive gastric/gastroesophagealcancer who received treatment with Drug A (10 mg/kg QW), trastuzumab,ramucirumab, and paclitaxel, (see Example 5B) two were not yetevaluable. One patient achieved PR based on investigator-assessedresponse using RECIST v1.1 criteria.

Among the three patients with HER2-positive gastric/gastroesophagealcancer who received treatment with Drug A (15 mg/kg QW), trastuzumab,ramucirumab, and paclitaxel, (see Example 5B) two were not yetevaluable. One patient achieved PR based on investigator-assessedresponse using RECIST v1.1 criteria. Low rates of cytopenias wereobserved.

Drug A in combination with pembrolizumab, 5-fluorouracil, and a platinumagent showed clinical activity in the treatment of advanced 1L HNSCC(i.e., as a first treatment in patients with advanced HNSCC who have notreceived prior therapy for HNSCC.) Drug A in combination withtrastuzumab, ramucirumab, and paclitaxel showed clinical activity in thetreatment of advanced≥2L gastric/gastroesophageal cancer (i.e., as atreatment in patients who have received at least one prior therapy forgastric or GEJ cancer).

Results from pharmacodynamics analyses indicated that near complete CD47target occupancy (also known as receptor occupancy) is maintainedthroughout the Drug A dosing interval when combined withchemotherapy-containing regimens.

Example 5D: Additional Results from the Exemplary Clinical TrialsDescribed in Example 5A

CD47 is a myeloid checkpoint up-regulated by tumors to evade theanticancer immune response. Drug A is an exemplary high affinityCD47-blocking fusion protein with an inactive Fc region designed tosafely enhance anticancer therapeutics (Kauder et al. (2018) PLoS ONE.13(8): e0201832: Chow et al. (2020) Journal of Clinical Oncology. 38:15suppl, 3056-3056). Drug A in combination with standard chemotherapy andantibody regimens was evaluated in patients with advanced HER2-positivegastric cancer (GC) or with head and neck squamous cell carcinoma(HNSCC).

Methods

Patients with previously treated advanced HER2-positive GC received DrugA (A) 10 mg/kg QW or 15 mg/kg QW in combination with trastuzumab(T)+ramucirumab (ram)+paclitaxel (pac) as 2nd or later-line treatment.The GC patients had progressed during or following a priorfluoropyrimidine therapy (or a fluoropyrimidine-containing therapy). GCpatients who had progressed during or following a prior therapy withtrastuzumab and/or a platinum-based chemotherapeutic agent wereincluded. Patients with untreated advanced HNSCC received Drug A (A) 10mg/kg QW or 15 mg/kg QW in combination with pembrolizumab(P)+5FU+platinum (cisplatin or carboplatin) as 1st line therapy. Theprimary endpoint was dose limiting toxicity (DLT). Tumor response,pharmacokinetic (PK), and pharmacodynamic (PD) markers were assessed inall patients.

Results

Fifty-five pts were enrolled in the study. Patients' baselinecharacteristics are shown in Table B:

TABLE B Baseline Characteristics Drug A + Drug A + trastuzumab +pembrolizumab + chemo chemo ≥2L GC (N = 14) 1L HNSCC (N = 5) Median age,years (range) 63 (36-83) 61 (45-63) Sex, n M 10 4 F  4 1 Race, n Asian11 4 White  3 1 Other — — ECOG PS, n 0  5 4 1  9 1 Progressed upon prior13 (93)  N/A anti-HER2 Therapy, n (%,) Progressed upon ≥2 prior  1 (7.1)N/A anti-HER2 therapy n (%) Progressed upon prior  1 (7.1) 0 (0)  CPITherapy, n (%) Visceral distant metastasis, 13 (93)  1 (20) n (%)

1 patients with ≥2L GC received A+T+ram+pac and were evaluated forsafety. No dose-limiting toxicities (DLTs) were reported, and the Drug Amaximum administered dose was 15 mg/kg QW. Of the 9 patients whoexperienced any adverse event, 8 patients reported treatment-relatedadverse events (TRAE). The most common TRAEs were low grade diarrhea,fatigue, pruritus/urticaria and rash (each n=21%). TRAEs≥Grade 3severity were of low frequency. There were no treatment related SAEsreported amongst GC patients treated with A+T+ram+pac. Among the 11 GCpatients who received Drug A at 15 mg/kgqw+trastuzumab+ramucirumab+paclitaxel, 7 demonstrated partial response,3 demonstrated stable disease, and 1 demonstrated progressive disease.Among the 3 patients who received Drug A at 10 mg/kgqw+trastuzumab+ramucirumab+paclitaxel, 2 demonstrated partial response,and one demonstrated stable disease.

Three patients with previously untreated HNSCC were administeredA+P+5FU+platinum, as described above. No DLTs were reported. Three ptsexperienced any adverse events (AE), none were treatment-related. TheHNSCC patient who received Drug A at 15 mg/kgqw+pembrolizumab+5-fluorouracil+a platinum-based chemotherapeutic agentwas CPI naive and demonstrated partial response. Of the three patientswho received Drug A at 10 mg/kg qw+pembrolizumab+5-fluorouracil+aplatinum-based chemotherapeutic agent, all were CPI naïve. One patientdemonstrated complete response, one patient demonstrated partialresponse, and one demonstrated progressive disease.

The clinical activity of Drug A chemotherapy combinations in responseevaluable patients are summarized in Table C below:

TABLE C Responses to Drug A Chemotherapy Combinations ORR Median Follow-Patient/Treatment N (95% CI) up* (95% CI) >2L Gastric Cancer/ 14 64.3%[38.8%; 5.3 Drug A + trastuzumab + 83.7%] [2.8; 6.7] ramucirumab +paclitaxel Drug A (15 mg/kg qw) 11 63.6% [35.4%; 4.2 84.8%] [2.4; 6.2]Drug A (10 mg/kg qw)  3 66.7% [20.8%; 8.9 93.9%] [5.1; 9.6] 1LHNSCC/Drug A +  4   75% [30.0%; 5.0 pembrolizumab + 5FU + 95.0%] [1.3;8.8] platinum Drug A (15 mg/kg qw)  1 100% [20.5; 1.6 100%] [1.3; 1.9]Drug A (10 mg/kg qw)  3 66.7% [20.8%; 5.3 93.9%] [5.0; 8.8]

Initial Drug A combination PK and CD47 target occupancy are similar tothat of single agent administration. Near complete (80%-100%) CD47target occupancy is maintained throughout Drug A dosing interval whencombined with chemotherapy-containing regimens. Circulating immune cellprofiles (CD4⁺ T cells, CD8⁺ T cells, CD19⁺ B cells, and CD16⁺ CD56⁺ NKcells) are generally unchanged following Drug A combined withchemotherapy-containing regimens. Drug A PK following combinationtherapies with pembrolizumab or trastuzumab is comparable, with andwithout chemotherapy.

CONCLUSIONS

Preliminary data indicated that Drug A is well tolerated and can besafety combined with the anticancer antibody+multi-agent chemotherapyregimens studied with no maximum tolerated dose reached. The maximumadministered dose of Drug A in combination was 15 mg/kg QW.

Drug A demonstrates initial ORR of 64% in patients with ≥2L HER2positive GC in combination with trastuzumab and ramucirumab+paclitaxelthat compares favorably with the clinical experience oframucirumab+paclitaxel in patients whose disease has progressed uponprior trastuzumab-containing regimens.

Drug A demonstrates initial anti-cancer activity including complete andpartial objective responses in combination withpembrolizumab+5FU+platinum in patients who have not received priortreatment for their advanced HNSCC.

Preliminary pharmacokinetics and pharmacodynamic analysis demonstratesno impact of the combination partners upon Drug A exposure levels withfull CD47 receptor occupancy.

Example 6: Further Characterization of Fusion Polypeptides Comprising aSIRPα Variant and an Fc Variant

Previous studies described in Liu et al. (2015) Nature Medicine. 21(1):1; Soto-Pantoja et al. (2014) Cancer Research. 74(23): 6771-83; andTseng D et al. (2013) Proc Natl Acad Sci USA. 110(27): 11103-11108 havethat shown dendritic cells (DCs) and T play an important role inantitumor response. The effects of the administration of Drug A, Drug B,or Drug C on DC activation were assessed in a mouse model. Drug A is afusion polypeptide comprising a SIRPα variant that binds hCD47 with aK_(D) of ˜140 pM. The C-terminus of the SIRPα variant of Drug A is fusedto the N-terminus of an Fc variant with ablated effector function. DrugB is a fusion polypeptide comprising the SIRPα variant of Drug A whoseC-terminus is fused to the N-terminus of a WT Fc (i.e., the WT Fc fromwhich the Fc variant of Drug A was derived). Drug C is a fusionpolypeptide comprising a SIRPα variant that binds hCD47 with a K_(D) of˜3 nM whose C-terminus is fused to the N-terminus of the Fc variant ofDrug A.

Briefly, C57BL6 mice were randomized into 4 groups (n=3 per group) andadministered with 3 mg/kg (or “mpk”) of Drug A, Drug B, or Drug C, orwith vehicle (PBS). 3.5 hours following intravenous injection, spleenswere harvested an analyzed for up-regulation of CD86, a cell-surfacemarker that indicates dendritic cell activation. As shown in FIGS. 6A,6B, 7A, and 7B, CD8⁺ and CD8⁻ dendritic cells were activated in spleensof mice that were administered with Drug A. The level of CD8⁺ dendriticcell activation (FIG. 6A) and CD8⁻ dendritic cell activation (FIG. 6B)in the spleens of mice administered with Drug C was the same as in miceadministered with PBS control. As shown in FIGS. 7A and 7B, CD8⁺ andCD8⁻ dendritic cells were activated in the spleens of mice administeredwith Drug B, but to a lesser extent than CD8⁺ and CD8⁻ dendritic cellsin the spleens of mice administered with Drug A. These data indicatethat the administration of a therapeutic agent comprising a CD47 bindingmoiety (e.g., a SIRPα variant) that has an affinity for hCD47 that isbetter than about 10 nM and/or an Fc variant with ablated effectorfunction leads to higher CD8⁺ and CD8⁻ DC activation than administrationof a therapeutic agent that comprises a CD47 binding moiety (e.g., aSIRPα variant) that has an affinity for CD47 (e.g., hCD47) that ishigher than 10 nM and/or a WT Fc domain. Moreover, a therapeutic agentthat binds CD47 and comprises an Fc domain with ablated effectorfunction (e.g., a fusion polypeptide described herein) demonstratesimproved safety following administration as compared to a therapeuticagent that binds CD47 and comprises a WT Fc domain. See, e.g., Kauder etal. (2018) PLoS ONE 13(8): e0201832.

In vitro receptor occupancy assays were performed to assess the bindingof Drug A, F59/magrolimab, TTI-621, and TTI-622 to hCD47. As discussedherein, Drug A is a fusion polypeptide comprising a SIRPα variant thatbinds hCD47 with a K_(D) of ˜140 pM whose C-terminus is fused to theN-terminus of an Fc variant with ablated effector function.F59/magrolimab is a therapeutic anti-CD47 antibody comprising a humanIgG4 Fc domain with WT effector function. TTI-621 is a therapeuticfusion polypeptide comprising the CD47 binding domain of human SIRPαlinked to a human IgG1 Fc domain with WT effector function. TTI-622 is atherapeutic fusion polypeptide comprising the CD47 binding domain ofhuman SIRPα linked to a to a human IgG4 Fc domain with WT effectorfunction. The affinities of Drug A, F59/magrolimab, TTI-621, and TTI-622for hCD47 are shown in Table D below.

TABLE D K_(D) for human Drug/Agent CD47 (nM) Drug A 0.14 nMF59/magrolimab 7 nM TTI-621 500 nM TTI-622 500 nM

As shown in FIG. 8A, Drug A exhibited about 100% receptor occupancy at aconcentration of ˜1 nM. F59/magrolimab exhibited about 90% receptoroccupancy at a concentration of ˜1 nM. Agents 2 and 3 exhibited about40% receptor occupancy at a concentration of ˜1 μM.

A validated SIRPα signaling assay (PathHunter SIRPα Signaling Bioassayfrom DiscoverX) was used to assess the degree to which Drug A,F59/magrolimab, TTI-621, and TTI-622 inhibit the interaction betweenhSIRPα and hCD47. The EC50 values of Drug A, F59/magrolimab, TTI-621,and TTI-622 for hCD47 are shown in Table E below.

TABLE E EC50 Drug/Agent (ng/ml) Drug A 25.1 F59/magrolimab 74.4TTI-621 >150 TTI-622 >150

A shown in FIG. 8B, at a concentration of 1 nM, Drug A completelyinhibited SIRPα signaling. F59/magrolimab inhibited SIRPα signaling byabout 80% at a concentration of 1 nM. By contrast, TTI-621 and TTI-622inhibited SIRPα signaling by less than about 10% at a concentration of 1nM.

The preceding Examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and fall within the scope of the appendedclaims.

1. A method of treating cancer in an individual, comprisingadministering to the individual an effective amount of: (a) apolypeptide comprising a SIRPα D1 domain variant and an Fc domainvariant, and (b) a Bcl-2 inhibitor; wherein the SIRPα D1 domain variantcomprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85;wherein the Fc domain variant is (i) a human IgG1 Fc region comprisingL234A, L235A, G237A, and N297A mutations, wherein numbering is accordingto the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S,P331S, and N297A mutations, wherein numbering is according to the EUindex of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P,F234V, L235A, and delG236 mutations, wherein numbering is according tothe EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P,E233P, F234V, L235A, delG236, and N297A mutations, wherein numbering isaccording to the EU index of Kabat. 2-7. (canceled)
 8. A method oftreating cancer in an individual, comprising administering to theindividual an effective amount of: (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, and (b) a platinum-basedchemotherapy agent; wherein the SIRPα D1 domain variant comprises theamino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fcdomain variant is (i) a human IgG1 Fc region comprising L234A, L235A,G237A, and N297A mutations, wherein numbering is according to the EUindex of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, andN297A mutations, wherein numbering is according to the EU index ofKabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V,L235A, and delG236 mutations, wherein numbering is according to the EUindex of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P,F234V, L235A, delG236, and N297A mutations, wherein numbering isaccording to the EU index of Kabat. 9-11. (canceled)
 12. A method oftreating cancer in an individual, comprising administering to theindividual an effective amount of: (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, (b) a PD-1 inhibitor, (c) anantimetabolite, and (d) a platinum-based chemotherapy agent; wherein theSIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a human IgG1Fc region comprising L234A, L235A, G237A, and N297A mutations, whereinnumbering is according to the EU index of Kabat; (ii) a human IgG2 Fcregion comprising A330S, P331S, and N297A mutations, wherein numberingis according to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat,wherein the cancer is head and neck squamous cell carcinoma (HNSCC), andwherein the individual has not received prior treatment for HNSCC. 13.The method of claim 12, wherein the HNSCC is advanced and/or metastaticHNSCC.
 14. The method of claim 12 or 13, wherein the PD-1 inhibitor isan anti-PD-1 antibody.
 15. The method of claim 14, wherein the anti-PD-1antibody is pembrolizumab, nivolumab, pidilizumab, cemiplimab, orBMS-936559.
 16. (canceled)
 17. The method of claim 12, wherein theantimetabolite is 5-fluorouracil, 6-mercaptopurine, capecitabine,cytarabine, floxuridine, fludarabine, gemcitabine, hydroxycarbamide,methotrexate, pemetrexed, phototrexate.
 18. (canceled)
 19. The method ofclaim 12, wherein the platinum-based chemotherapy agent is carboplatin,cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate,phenanthriplatin, picoplatin, or satraplatin. 20-21. (canceled)
 22. Amethod of treating cancer in an individual, comprising administering tothe individual an effective amount of: (a) a polypeptide comprising aSIRPα D1 domain variant and an Fc domain variant, (b) an anti-HER2antibody, and (c) an anti-PD-L1 antibody; wherein the SIRPα D1 domainvariant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat.23-28. (canceled)
 29. A method of treating cancer in an individual,comprising administering to the individual an effective amount of: (a) apolypeptide comprising a SIRPα D1 domain variant and an Fc domainvariant, (b) an anti-HER2 antibody, (c) an anti-VEGF2 antibody, and (d)paclitaxel; wherein the SIRPα D1 domain variant comprises the amino acidsequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domainvariant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A,and N297A mutations, wherein numbering is according to the EU index ofKabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297Amutations, wherein numbering is according to the EU index of Kabat;(iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, anddelG236 mutations, wherein numbering is according to the EU index ofKabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V,L235A, delG236, and N297A mutations, wherein numbering is according tothe EU index of Kabat, wherein the cancer is gastric cancer orgastroesophageal junction (GEJ) cancer, and wherein the individual hasreceived at least one prior therapy for the gastric or the GEJ cancer.30. The method of claim 29, wherein the individual has received priortherapy with an anti-HER2 antibody, with an anti-HER2 antibody and afluoropyrimidine, or with an anti HER2 antibody and a platinum-basedchemotherapy agent.
 31. The method of claim 29, wherein the anti-HER2antibody is trastuzumab.
 32. The method of claim 29, wherein theanti-VEGF antibody is ramucirumab.
 33. The method of claim 29, whereinthe gastric cancer or the GEJ cancer is HER2⁺ gastric cancer or HER2⁺GEJ cancer.
 34. The method of claim 12 wherein the polypeptidecomprising a SIRPα D1 domain variant and an Fc domain variant isadministered at a dose of 10 mg/kg once a week or at a dose of 15 mg/kgonce a week.
 35. (canceled)
 36. A method of treating cancer in anindividual, comprising administering to the individual an effectiveamount of (a) a polypeptide comprising a SIRPα D1 domain variant and anFc domain variant, and (b) an anti-TROP2 antibody; wherein the SIRPα D1domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat.37-39. (canceled)
 40. The method of claim 12, wherein the Fc domainvariant is a human IgG1 Fc region comprising L234A, L235A, G237A, andN297A mutations, wherein numbering is according to the EU index ofKabat.
 41. The method of claim 40, wherein the Fc domain variantcomprises the amino acid sequence of SEQ ID NO:
 91. 42. The method ofclaim 12, wherein the polypeptide comprising a SIRPα D1 domain variantand an Fc domain variant comprises the amino acid sequence of SEQ ID NO:135 or
 136. 43. (canceled)
 44. The method of claim 12, wherein thepolypeptide comprising a SIRPα D1 domain variant and an Fc domainvariant forms a homodimer.
 45. The method of claim 12, wherein theindividual is a human. 46-75. (canceled)